In today’s data-driven landscape, the efficient transfer of large datasets to and from <a href="https://aws.amazon.com/s3/">Amazon Simple Storage Service</a> (Amazon S3) is a critical piece of an enterprise’s cloud strategy
Common business use cases that need frequent transferring of large data sets include cloud-based data lakes that depend on receiving data from various sources
Amazon S3 can also serve as the starting point for your Generative AI journey
Generative AI applications need large data sets
and by transferring this data into Amazon S3 organizations can use the full suite of Amazon Web Services (AWS) artificial intelligence/machine learning (AI/ML) tools
Once a model is trained in AWS using this data
the model artifacts can also be stored in Amazon S3
Other use cases include backup and restore
There are three general patterns when transferring data to and from Amazon S3:
This post details three network architectures for setting up connectivity for the pattern using AWS Direct Connect
These architectures differ in terms of the services used
Understanding these network design options and their tradeoffs is crucial for organizations to optimize their cloud storage operations
The following services are included in the architectures that are covered
The following are brief descriptions of each one
You can select the links to learn more about each service
All three of the architectures that are covered use Direct Connect
then you can configure a new VIF on the existing connection
If you have a hosted connection that only supports one VIF
then you must order an additional hosted connection to support an additional VIF
If your plan is to have a landing zone with many VPCs and to provide access to AWS services and applications inside and outside of those VPCs, then a dedicated connection is recommended because it provides more flexibility with the network design. It is also recommended to have at least two connections for resiliency as covered in&nbsp;the <a href="https://docs.aws.amazon.com/directconnect/latest/UserGuide/resiliency_toolkit.html">AWS Direct Connect Resiliency Toolkit</a>
The Direct Connect charges include a port hours charge that is based on the type of connection and the capacity of the connection. There is also a charge based on how much data is transferred outbound from AWS to on-premises. Data that is transferred inbound from on-premises to AWS is free. For more details on pricing, refer to the <a href="https://aws.amazon.com/directconnect/pricing/">Direct Connect pricing</a>&nbsp;page
Each architecture description includes a pricing estimate based on the following example scenario
You have two 10 Gbps dedicated Direct Connect connections and you want to setup connectivity to Amazon S3 for data transfer from on-premises
You transfer an estimated 4 TB of data per month into Amazon S3
You estimate that you must retrieve 2 TB of data per month from Amazon S3 and transfer it back to on-premises
The following calculations are based on the <a href="https://calculator.aws/#/">AWS Pricing Calculator</a> that can be used to run your own calculations based on your architecture and specific use case
While all pricing shown in the following architecture examples is based on AWS Regions in the United States
The AWS Pricing Calculator can be used to show pricing information for these other AWS Regions
The following figure shows this architecture using two Direct Connect connections
each with a public VIF connecting to the AWS network
This architecture is a good choice if you want to minimize the data transfer costs related to transferring data into Amazon S3
it needs additional configuration for the public VIF
because it exposes the on-premises network to the AWS public network
thus you need to take this into consideration
the costs associated with this architecture include the Direct Connect charges as shown in the following table
The total estimated charges would be $6,610.96 USD a month
the traffic is sent to Amazon S3 over the AWS network
each with a Private VIF connecting through the Direct Connect Gateway to a VPC with an interface endpoint
Figure 2: Private VIF through VPC interface endpoint
This architecture is a good choice if you do not want to create a public VIF
You may not want to add the additional configuration needed to filter the advertised Amazon prefixes
You may have security concerns with connecting to the AWS public network
especially if appropriate security services are not in-line with the Direct Connect connection
You may already have a hosted connection with a Private VIF that you prefer to use rather than ordering another hosted connection for another VIF
The costs associated with this architecture include the Direct Connect charges covered in Architecture 1 in addition to the interface endpoint charges shown in the following table
The total estimated charges would be $6,610.96 USD + $90.64 USD = $6,701.60 USD per month
This architecture uses a Transit VIF and the Transit Gateway
A single Transit VIF can be used to connect to all VPCs through the Transit Gateway
This avoids having to configure a separate Private VIF for each VPC
which was common practice before the Transit Gateway service became available
In this architecture a VPC with an interface endpoint is attached to the Transit Gateway
which is connected back to on-premises using a Transit VIF
Access to Amazon S3 through the interface endpoint would work as described in Architecture 2
with the exception being that traffic would flow over the Transit VIF to the interface endpoint
each with a Transit VIF connecting through the Direct Connect Gateway to the same Transit Gateway that connects to a VPC with an interface endpoint
Figure 3: Transit VIF through Transit Gateway and VPC interface endpoint
If you already have a Transit Gateway in place
then this architecture is the least complex from an implementation and operational standpoint and the most scalable
It is a good choice if you do not want to create additional VIFs or already have a hosted connection with a VIF and do not want to order another hosted connection for another VIF
It allows you to use a single Transit VIF to send data to VPCs and to Amazon S3 without configuring additional VIFs
It requires configuring the Transit Gateway and all of the attachments along with Transit Gateway routing tables
The costs associated with this architecture include the Direct Connect charges and interface endpoint charges covered in Architectures 1 and 2
in addition to the Transit Gateway charges as shown in the following table
The total estimated charges would be $6,610.98 USD + $90.64 USD + $318.76 USD= $7,020.38 USD per month
This post discussed different network architectures for transferring large datasets between on-premises environments and Amazon S3 using AWS Direct Connect
By understanding these architecture options
and Transit VIFs through the Transit Gateway
in addition to the implications of using each service from a cost and configuration complexity standpoint
you can choose the right design for your organization
The choice is influenced by a number of things
If you are a new user that is building a landing zone for the first time, then you can reference the&nbsp;<a href="https://docs.aws.amazon.com/whitepapers/latest/hybrid-connectivity/hybrid-connectivity-type-and-design-considerations.html">Hybrid Network Connectivity Whitepaper</a>&nbsp;to learn more about the considerations for choosing the right connectivity type and connectivity design
2024: An earlier version of this post incorrectly stated that AWS IP address ranges from ip-ranges.json can be used for BGP prefix filtering
Architecture 3 has been updated to include multiple interface endpoints for a more resilient architecture
The amount of outbound traffic has been adjusted to reflect a more realistic example.&nbsp;
Chuck is a Senior Solutions Architect at AWS with a background in Network Engineering
he works with customers to design and build innovative
resilient and cost-effective solutions in the AWS Cloud ensuring success in their cloud adoption journeys
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HIV-1 has well-established mechanisms to disrupt essential pathways in people with HIV
diversity of the amino acid sequences in fundamental HIV-1 proteins including Tat and Vif
have been linked to dysregulating these pathways
and subsequently influencing clinical outcomes in people with HIV
the relationship between Tat and Vif amino acid sequence variation and specific immune markers and metabolites of the tryptophan-kynurenine (Trp-Kyn) pathway remains unclear
this study aimed to investigate the relationship between Tat/Vif amino acid sequence diversity and Trp-Kyn metabolites (quinolinic acid (QUIN)
as well as specific immune markers (sCD163
NGAL and hsCRP) in n = 67 South African cART-naïve people with HIV
Sanger sequencing was used to determine blood-derived Tat/Vif amino acid sequence diversity
a LC–MS/MS metabolomics platform was employed using a targeted approach
Enzyme-linked immunosorbent assays and the Particle-enhanced turbidimetric assay was used
sCD163 (p = 0.042) and KA (p = 0.031) were higher in participants with Tat signatures N24 and R57
and amino acid variation at position 24 (adj R2 = 0.048
p = 0.031) of Tat were associated with sCD163 and KA
These preliminary findings suggest that amino acid variation in Tat may have an influence on underlying pathogenic HIV-1 mechanisms and therefore
this line of work merits further investigation
previous studies highlight that these specific amino acid signatures may be important in the pathogenesis of HIV-1
there is a need to profile the influence of viral protein amino acid sequence diversity in subtype C-specific cohorts
we aimed to use an exploratory approach to investigate the potential associations between specific Tat and Vif amino acid sequence variants with soluble urokinase plasminogen activator receptor (suPAR)
high-sensitivity C-reactive protein (hsCRP)
soluble CD163 (sCD163) and neutrophil gelatinase-associated lipocalin (NGAL)
as well as Trp-Kyn pathway metabolites in a cART treatment-naive South African cohort
all participants received counselling from a trained counsellor
The initial step in determining their HIV status involved using the First Response rapid HIV card test (Premier Medical Corporation Limited
following the protocol outlined by the South African Department of Health
the SD BIOLINE HIV 1/2 3.0 card test (Standard Diagnostics
they then received post-counselling and were referred to the nearest clinical/hospital for further assessment
CD4 + counts were analysed using the flow cytometric method (Beckman COULTER EPICS XL™ machine
whole blood samples were collected in EDTA tubes
samples underwent centrifugation at 2000 × g for 15 min at 10 °C within a 2-h timeframe
the samples were transferred into microcentrifuge tubes
and subsequently stored at -80 °C until further analysis
the same rapid freezing process was employed
but they were maintained at -18 °C for a maximum of five days before transportation to the laboratory
these samples were again stored at -80 °C until they underwent subsequent analysis
it is reasonable to hypothesize that Tat and Vif amino acid variations may influence other immune markers
Plasma hsCRP were analysed using a particle-enhanced turbidimetric assay (Cobas Integra 400 plus
while IL-6 levels were determined via the electrochemiluminescence immunoassay method (Elecsys 2010
ELISA assays (R&amp;D Systems DuoSet) were employed for plasma sCD163 and NGAL measurements
following the instructions from the manufacturer
The coefficients of variation for both intra- and inter-assay tests fell within acceptable ranges
Key mutations within the Tat/Vif regions were identified and specifically highlighted for further analysis
All analyses were carried out utilizing SPSS software (IBM
P-values below 0.05 were deemed statistically significant for all analyses
Normality of variables was evaluated by visually inspecting QQ plots alongside descriptive statistics
It was observed that the data distribution of immune markers IL-6
the data for skewed variables were log-transformed prior to statistical analyses
Data presented acceptable skewness and kurtosis values within the range of -2 and 2
residual plots indicated homoscedasticity and linearity
The Durbin-Watson statistic was within acceptable range
residuals of the regression models were normally distributed
χ2 tests were utilized to assess group disparities across amino acid variants for sex
Independent sample T-tests were utilized to detect differences in study characteristics (such as age
as well as levels of immune markers/metabolites
For the χ2 tests and independent sample t-tests
p values of &lt; 0.05 we deemed significant
To correct for the number of immune markers or metabolites tested
a Bonferroni correction was implemented (α/n = 0.05/5 = 0.01) in all relevant analyses
Pearson correlation analysis was utilized to identify covariates by exploring correlations between sociodemographic and lifestyle variables (age
and locality) and specific immune markers/metabolites
An Analysis of Covariance (ANCOVA) was used to adjust for the influence of covariates
which helps in isolating the effect of the independent variable on the dependent variable
with immune marker/metabolite levels as the dependent variables
to compare their levels among Tat or Vif amino acid variants
and locality in the investigation of immune markers
and for alcohol use and BMI in the examination of metabolites to prevent model overfitting
Multiple regression analysis using the enter method was employed to determine associations between Tat/Vif amino acid variants and immune marker/metabolite levels after adjusting for covariates
For the Pearson correlation and multiple regression analyses
Participants were stratified based on Tat amino acid variants at position 24 (K: 14 vs
as well as Vif amino acid variants at position 17 (K: 25 vs
Using independent sample t-tests as well as χ2 tests
no significant differences were found in study characteristics (sex
and smoking) amongst the investigated groups
Participants with the Tat N24 variant had higher levels of sCD163 compared to participants with the K24 variant (p = 0.04) (supplementary Fig
sCD163 levels were significantly higher in participants with Tat S31 compared to participants with C31 (p &lt; 0.001) (supplementary Fig
following the application of a Bonferroni correction (p = 0.05/5 = 0.01)
only higher levels of sCD163 remained statistically significant in participants with the S31 variant (p &lt; 0.001)
None of the remaining Tat amino acid variants (positions 24
and 68) displayed significant differences for any of the immune markers or metabolites investigated (supplementary Figs
None of the immune markers or metabolites were significantly different between the Vif amino acid variants (supplementary Figs
</a>Significant findings for Tat position 24 and 57
(A) sCD163 levels were significantly higher in participants with the N24 amino acid variant in contrast to participants with the K24 amino acid variant (p = 0.042)
KA levels were significantly higher in the participants with the R57 amino acid variant compared to participants with the S57 variant (p = 0.031)
The bars depict the average protein concentrations across the diverse study groups and are articulated as mean values with standard error of the mean (SEM)
</a>Volcano plot demonstrating the association between viral protein amino acid variations and immune marker/metabolite levels. The plot includes Tat amino acid position 24 (blue circles), position 31 (brown circles), position 57 (green circles), and position 68 (black circles). It also includes Vif amino acid positions 17 (orange triangles) and 31 (yellow triangles). Significant values, those with &gt; -log10 1.3, are in red text
</a>Heatmap representing the associations (normalized effect size β) between Tat/Vif amino acid positions and peripheral immune and metabolic markers
Significant associations with p-value &lt; 0.05 are indicated by white asterisks
Several key findings emerged: (1) After adjusting for covariates
sCD163 and KA were higher in participants with Tat signatures N24 and R57
respectively and (2) amino acid variation at position 24 and 57 of Tat were associated with sCD163 and KA
this previous study also investigated a small cohort; therefore
these findings warrant further validation in larger cohorts
further investigation is necessary to elucidate the complete functional significance of variation at this position
Here our results also showed that participants with the R57 signature had higher levels of KA
and amino acid variation at this position (between R57 and S57) was associated with KA
a consistent trend for the impact of the R57 variant is observed in the dysregulation of metabolism
indicating the necessity for larger cohort studies to further investigate this amino acid signature
This implies that the Tat C31S status might not serve as an adequate biomarker for adverse clinical outcomes
as the effects of mutations at this position could be concealed by other unexplored clinical factors
no significant findings were reported for the influence of these amino acid signatures
The precise roles of these Tat and Vif variants are yet to be fully explored
it is evident from this study and many others that amino acid sequence variations of viral proteins may influence the structure–function relationships of these proteins
ultimately impacting the underlying mechanisms of HIV-1 pathogenesis
the extent to which these variations contribute to overall clinical outcomes in people with HIV requires further investigation and cannot be fully ascertained from this study alone
We acknowledge that certain signatures investigated in cell culture (without confounders) similarly reflect characteristics in clinical sample types
This discrepancy may stem from the fact that in people with HIV
several other proteins may influence the levels of these markers
or there may be other confounding factors at play in clinical investigations
it is worth considering that certain amino acids may not directly influence outcomes
their positioning in critical functional regions of key viral proteins may lead to changes in underlying mechanisms
there may be additional potential covariates that could have influenced our findings in the clinical sample
we have adjusted our analysis based on demographics and study characteristics that we have determined to have an influence
Another limitation of our study is related to the number of statistical tests performed and the multiple testing correction strategy employed
To identify covariates associated with our outcomes of interest
This approach allowed us to adjust for only those covariates that were significantly associated
thereby reducing the risk of overfitting the model
While this conservative approach helps control the family-wise error rate
it also increases the likelihood of Type II errors
which means some true associations may not have been detected
The stringent nature of the Bonferroni correction can lead to an overly cautious interpretation of results
potentially overlooking meaningful findings
we only investigated specific immune markers and metabolites
and there may be other markers more directly involved in pathways related to the amino acid changes of the proteins we investigated
the exploratory nature of this study should be taken into consideration when interpreting the findings presented here
In a treatment-naïve cohort from South Africa subtype C
we investigated the associations between changes in amino acid sequences in Tat and Vif and specific immune and metabolic markers
respectively and amino acid variation at position 24 and 57 of Tat were associated with sCD163 and KA
Findings from this study highlight the potential influence of amino acid sequence variation of the Tat protein on inflammatory and metabolic pathways in people with HIV
The data supporting the findings of this study are available in the supplementary material of this article
The sequences are accessible in GenBank under the accession numbers OR621303-OR621349 for Tat and OR194556-OR194606 for Vif
UNAIDS. In danger: UNAIDS global AIDS update 2022. Joint United Nations Programme on HIV/AIDS; 2022. Available from: <a href="https://www.unaids.org/en/resources/documents/2022/in-danger-global-aids-update" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="https://www.unaids.org/en/resources/documents/2022/in-danger-global-aids-update">https://www.unaids.org/en/resources/documents/2022/in-danger-global-aids-update</a>
The epidemic of HIV/AIDS in developing countries; the current scenario in Pakistan
The influence of viral protein R amino acid substitutions on clinical outcomes in people living with HIV: A systematic review
Variation in HIV-1 Nef function within and among viral subtypes reveals genetically separable antagonism of SERINC3 and SERINC5
Structural similarity-based predictions of protein interactions between HIV-1 and Homo sapiens
Molecular Modeling of Subtype-Specific Tat Protein Signatures to Predict Tat-TAR Interactions That May Be Involved in HIV-Associated Neurocognitive Disorders
Signatures of HIV-1 subtype B and C Tat proteins and their effects in the neuropathogenesis of HIV-associated neurocognitive impairments
HIV-1 integrase variants retarget viral integration and are associated with disease progression in a chronic infection cohort
HIV-1 Subtype C Vpr Amino Acid Residue 45Y and Specific Conserved Fragments Are Associated with Neurocognitive Impairment and Markers of Viral Load
New insights into pathogenesis point to HIV-1 Tat as a key vaccine target
Viral infectivity factor: a novel therapeutic strategy to block HIV-1 replication
Mutations in the highly conserved SLQYLA motif of Vif in a simian–human immunodeficiency virus result in a less pathogenic virus and are associated with G-to-A mutations in the viral genome
Targeting Tat–TAR RNA Interaction for HIV-1 Inhibition
The structural basis for HIV-1 Vif antagonism of human APOBEC3G
Antiretroviral therapy does not block the secretion of the human immunodeficiency virus tat protein
Partially active HIV-1 Vif alleles facilitate viral escape from specific antiretrovirals
An investigation of the HIV Tat C31S and R57S mutation on peripheral immune marker levels in South African participants: A pilot study
HIV-1 subtype C Tat exon-1 amino acid residue 24K is a signature for neurocognitive impairment
Differential effects of HIV type 1 clade B and clade C Tat protein on expression of proinflammatory and antiinflammatory cytokines by primary monocytes
Human immunodeficiency virus type 1 subtype C Tat fails to induce intracellular calcium flux and induces reduced tumor necrosis factor production from monocytes
Differential induction of interleukin-10 in monocytes by HIV-1 clade B and clade C Tat proteins
Impact of subtype C-specific amino acid variants on HIV-1 Tat-TAR interaction: insights from molecular modelling and dynamics
HIV-1 Tat amino acid residues that influence Tat-TAR binding affinity: a scoping review
A Naturally Occurring Polymorphism in the HIV-1 Tat Basic Domain Inhibits Uptake by Bystander Cells and Leads to Reduced Neuroinflammation
Differential anti-APOBEC3G activity of HIV-1 Vif proteins derived from different subtypes
Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest
HIV-1 Vif Triggers Cell Cycle Arrest by Degrading Cellular PPP2R5 Phospho-regulators
A Pilot Investigation of the Association Between Vpr Amino Acid Substitutions and Peripheral Immune Marker Levels in People With Human Immunodeficiency Virus: Implications for Neurocognitive Impairment
A pilot investigation of the association between HIV-1 Vpr amino acid sequence diversity and the tryptophan-kynurenine pathway as a potential mechanism for neurocognitive impairment
HIV-1 Vif protein sequence variations in South African people living with HIV and their influence on Vif-APOBEC3G interaction
Understanding the mechanisms driving the spread of subtype C HIV-1
Genetic and functional characterization of HIV-1 Vif on APOBEC3G degradation: First report of emergence of B/C recombinants from North India
HIV-Related Immune Activation and Inflammation: Current Understanding and Strategies
Persistent metabolic changes in HIV-infected patients during the first year of combination antiretroviral therapy
Clinical Relevance of Kynurenine Pathway in HIV/AIDS: An Immune Checkpoint at the Crossroads of Metabolism and Inflammation
Serum kynurenine-to-tryptophan ratio increases with progressive disease in HIV-infected patients
Associations among peripheral and central kynurenine pathway metabolites and inflammation in depression
The Prospective Urban Rural Epidemiology (PURE) study: Examining the impact of societal influences on chronic noncommunicable diseases in low-
The Plasma [Kynurenine]/[Tryptophan] Ratio and Indoleamine 2,3-Dioxygenase: Time for Appraisal
High-sensitivity C-reactive protein among people living with HIV on highly active antiretroviral therapy: a systemic review and meta-analysis
Plasma Soluble CD163 Level Independently Predicts All-Cause Mortality in HIV-1–Infected Individuals
Factors Associated With Plasma IL-6 Levels During HIV Infection
Soluble Urokinase Plasminogen Activator Receptor Is Predictive of Non-AIDS Events During Antiretroviral Therapy-mediated Viral Suppression
The Association of Immune Markers with Cognitive Performance in South African HIV-Positive Patients
The association of peripheral immune markers with brain cortical thickness and surface area in South African people living with HIV
ExPASy: The proteomics server for in-depth protein knowledge and analysis
Tryptophan metabolism and its relationship with central nervous system toxicity in people living with HIV switching from efavirenz to dolutegravir
The effects of highly active antiretroviral therapy on the serum levels of pro-inflammatory and anti-inflammatory cytokines in HIV infected subjects
sCD163 and sCD14 Levels Have Distinct Associations with Antiretroviral Treatment and Cardiovascular Disease Risk Factors
High soluble CD163 levels correlate with disease progression and inflammation in Kenyan children with perinatal HIV-infection
Interleukin 6 Is a Stronger Predictor of Clinical Events Than High-Sensitivity C-Reactive Protein or D-Dimer During HIV Infection
Kynurenic Acid: The Janus-Faced Role of an Immunomodulatory Tryptophan Metabolite and Its Link to Pathological Conditions
Tat protein of human immunodeficiency virus type 1 subtype C strains is a defective chemokine
Clade C HIV-1 isolates circulating in Southern Africa exhibit a greater frequency of dicysteine motif-containing Tat variants than those in Southeast Asia and cause increased neurovirulence
NMDA receptor activation by HIV-Tat protein is clade dependent
Impact of the HIV Tat C30C31S dicysteine substitution on neuropsychological function in patients with clade C disease
Neuroimaging abnormalities in clade C HIV are independent of Tat genetic diversity
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Open access funding provided by North-West University
MEW received funding from the NRF Thuthuka grant (TTK22031652) and the Poliomyelitis Research Foundation (PRF) grant (23/84)
LKA was supported by the NRF Postgraduate Scholarship (140524) and the PRF grant (23/24)
Hypertension in Africa Research Team (HART)
South African Medical Research Council Unit for Hypertension and Cardiovascular Disease
EJVV and ZL: Consulted on statistical analysis
The study protocol was approved by the Health Research Ethics Committee of North-West University
in compliance with the Declaration of Helsinki
All participants provided informed consent
Participants were provided with detailed information about the study objectives
They were informed that participation was voluntary
and they could withdraw from the study at any time without any negative consequences
Confidentiality and anonymity of the participants' data were assured throughout the study
Written informed consent was obtained from each participant
and copies of the consent forms are securely stored at the North-West University
The authors declare no competing interests
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
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HIV-1 Vif recruits host cullin-RING-E3 ubiquitin ligase and CBFβ to degrade the cellular APOBEC3 antiviral proteins through diverse interactions
Recent evidence has shown that Vif also degrades the regulatory subunits PPP2R5(A–E) of cellular protein phosphatase 2A to induce G2/M cell cycle arrest
As PPP2R5 proteins bear no functional or structural resemblance to A3s
it is unclear how Vif can recognize different sets of proteins
Here we report the cryogenic-electron microscopy structure of PPP2R5A in complex with HIV-1 Vif–CBFβ–elongin B–elongin C at 3.58 Å resolution
The structure shows PPP2R5A binds across the Vif molecule
with biochemical and cellular studies confirming a distinct Vif–PPP2R5A interface that partially overlaps with those for A3s
Vif also blocks a canonical PPP2R5A substrate-binding site
indicating that it suppresses the phosphatase activities through both degradation-dependent and degradation-independent mechanisms
Our work identifies critical Vif motifs regulating the recognition of diverse A3 and PPP2R5A substrates
whereby disruption of these host–virus protein interactions could serve as potential targets for HIV-1 therapeutics
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Multiple APOBEC3 restriction factors for HIV-1 and one Vif to rule them all
Multifaceted HIV-1 Vif interactions with human E3 ubiquitin ligase and APOBEC3s
Demystifying cell cycle arrest by HIV-1 Vif
Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein
APOBEC3G restricts HIV-1 to a greater extent than APOBEC3F and APOBEC3DE in human primary CD4+ T cells and macrophages
APOBEC3 proteins can copackage and comutate HIV-1 genomes
and APOBEC3H demonstrate a conserved capacity to restrict Vif-deficient HIV-1
Endogenous origins of HIV-1 G-to-A hypermutation and restriction in the nonpermissive T cell line CEM2n
The AID/APOBEC family of nucleic acid mutators
DNA deamination mediates innate immunity to retroviral infection
Hypermutation of HIV-1 DNA in the absence of the Vif protein
Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts
Retroviral restriction factor APOBEC3G delays the initiation of DNA synthesis by HIV-1 reverse transcriptase
APOBEC3G inhibits elongation of HIV-1 reverse transcripts
APOBEC3F can inhibit the accumulation of HIV-1 reverse transcription products in the absence of hypermutation
Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G
APOBEC3G inhibits DNA strand transfer during HIV-1 reverse transcription
Cytidine deaminases APOBEC3G and APOBEC3F interact with human immunodeficiency virus type 1 integrase and inhibit proviral DNA formation
Human immunodeficiency virus type 1 cDNAs produced in the presence of APOBEC3G exhibit defects in plus-strand DNA transfer and integration
APOBEC3F and APOBEC3G inhibit HIV-1 DNA integration by different mechanisms
Antiviral function of APOBEC3G can be dissociated from cytidine deaminase activity
Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted antiviral functions of APOBEC3G
Identification of APOBEC3DE as another antiretroviral factor from the human APOBEC family
HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation
Vif overcomes the innate antiviral activity of APOBEC3G by promoting its degradation in the ubiquitin-proteasome pathway
Adaptive evolution and antiviral activity of the conserved mammalian cytidine deaminase APOBEC3H
The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif
Ubiquitination of APOBEC3 proteins by the Vif-Cullin5-ElonginB-ElonginC complex
HIV-1 Vif blocks the antiviral activity of APOBEC3G by impairing both its translation and intracellular stability
is suppressed by the HIV-1 and HIV-2 Vif proteins
Induction of APOBEC3G ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex
Human APOBEC3F is another host factor that blocks human immunodeficiency virus type 1 replication
Vif hijacks CBF-beta to degrade APOBEC3G and promote HIV-1 infection
T-cell differentiation factor CBF-beta regulates HIV-1 Vif-mediated evasion of host restriction
The Vif and Vpr accessory proteins independently cause HIV-1-induced T cell cytopathicity and cell cycle arrest
The Vif accessory protein alters the cell cycle of human immunodeficiency virus type 1 infected cells
Greenwood, E. J. et al. Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants. eLife <a href="https://doi.org/10.7554/eLife.18296" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="10.7554/eLife.18296">https://doi.org/10.7554/eLife.18296</a> (2016)
Naamati, A. et al. Functional proteomic atlas of HIV infection in primary human CD4+ T cells. eLife <a href="https://doi.org/10.7554/eLife.41431" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="10.7554/eLife.41431">https://doi.org/10.7554/eLife.41431</a> (2019)
Human immunodeficiency virus type 1 Vif induces cell cycle delay via recruitment of the same E3 ubiquitin ligase complex that targets APOBEC3 proteins for degradation
Vif-CBFbeta interaction is essential for Vif-induced cell cycle arrest
The Ebola virus nucleoprotein recruits the host PP2A-B56 phosphatase to activate transcriptional support activity of VP30
Structural basis of host protein hijacking in human T-cell leukemia virus integration
B’-protein phosphatase 2A is a functional binding partner of delta-retroviral integrase
Moura, M. &amp; Conde, C. Phosphatases in mitosis: roles and regulation. Biomolecules <a href="https://doi.org/10.3390/biom9020055" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="10.3390/biom9020055">https://doi.org/10.3390/biom9020055</a> (2019)
Protein phosphatases in the regulation of mitosis
The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm
Crystal structure of a PP2A B56-BubR1 complex and its implications for PP2A substrate recruitment and localization
Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling
Marelli, S. et al. Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest. eLife <a href="https://doi.org/10.7554/eLife.53036" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="10.7554/eLife.53036">https://doi.org/10.7554/eLife.53036</a> (2020)
Critical role of PP2A-B56 family protein degradation in HIV-1 Vif mediated G2 cell cycle arrest
HIV-1 Vif triggers cell cycle arrest by degrading cellular PPP2R5 phospho-regulators
Salamango, D. J. et al. Functional and structural insights into a Vif/PPP2R5 complex elucidated using patient HIV-1 isolates and computational modeling. J. Virol. <a href="https://doi.org/10.1128/JVI.00631-20" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="10.1128/JVI.00631-20">https://doi.org/10.1128/JVI.00631-20</a> (2020)
HIV-1 viral infectivity factor interacts with TP53 to induce G2 cell cycle arrest and positively regulate viral replication
A conserved motif provides binding specificity to the PP2A-B56 phosphatase
Formation of stable attachments between kinetochores and microtubules depends on the B56-PP2A phosphatase
The PP2A(B56) phosphatase promotes the association of Cdc20 with APC/C in mitosis
Structural basis of antagonism of human APOBEC3F by HIV-1 Vif
Structural basis for HIV-1 antagonism of host APOBEC3G via Cullin E3 ligase
Distinct domains within APOBEC3G and APOBEC3F interact with separate regions of human immunodeficiency virus type 1 Vif
Identification of two distinct human immunodeficiency virus type 1 Vif determinants critical for interactions with human APOBEC3G and APOBEC3F
Identification of the HIV-1 Vif and human APOBEC3G protein interface
Structural basis for hijacking CBF-beta and CUL5 E3 ligase complex by HIV-1 Vif
Expanding the PP2A interactome by defining a B56-specific SLiM
Selective PP2A enhancement through biased heterotrimer stabilization
Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme
Highly accurate protein structure prediction with AlphaFold
Critical role for protein phosphatase 2A heterotrimers in mammalian cell survival
Nuclear export and centrosome targeting of the protein phosphatase 2A subunit B56alpha: role of B56alpha in nuclear export of the catalytic subunit
Evolutionarily conserved pressure for the existence of distinct G2/M cell cycle arrest and A3H inactivation functions in HIV-1 Vif
DeepEMhancer: a deep learning solution for cryo-EM volume post-processing
Crystal structures of APOBEC3G N-domain alone and its complex with DNA
Automated electron microscope tomography using robust prediction of specimen movements
cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination
UCSF Chimera–a visualization system for exploratory research and analysis
New tools for the analysis and validation of cryo-EM maps and atomic models
PHENIX: a comprehensive Python-based system for macromolecular structure solution
The PyMOL Molecular Graphics System (DeLano Scientific
UCSF ChimeraX: structure visualization for researchers
<a data-track="click" data-track-action="download citation references" data-track-label="link" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/s41594-024-01314-6?format=refman&amp;flavour=references">Download references</a>
Wang at the Brookhaven Laboratory Cryo-EM facility for assistance with data collection
We thank other Xiong laboratory members for discussions
This work was supported by National Institutes of Health (NIH) grant no
This work was supported in part by the Intramural Research Program of the NIH
Center for Cancer Research and by the Innovation Award
These authors contributed equally: Yingxia Hu
Department of Molecular Biophysics and Biochemistry
performed the biophysical and biochemical experiments
contributed to experiments and discussions
Nature Structural &amp; Molecular Biology thanks Christopher Hill and the other
reviewer(s) for their contribution to the peer review of this work
Primary Handling Editor: Katarzyna Ciazynska
in collaboration with the Nature Structural &amp; Molecular Biology team
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
Size exclusion chromatography (SEC) profiles and SDS-PAGE analysis of individual PPP2R5A
Vif/CBFβ/Cul5 E3 and their complexes (repeated twice independently)
The cryo-EM reconstructions (surface with fitted models in cartoon representation) of the fully assembled Vif/CBFβ/Cul5 E3/PPP2R5A showed no presence of PPP2R5A
The SEC profile and SDS-PAGE analysis of PPP2R5A/VCBC complex crosslinked by Bissulfosuccinimidyl suberate (BS3)
The large scale crosslinking of the complex has been repeated 7 times independently
The OD280 and OD260 are shown in blue and red
The protein bands were detected by Coomassie blue stain
<a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41594-024-01314-6#MOESM7">Source data</a>
The Vif flexible loop that shows the largest local conformational changes upon A3F or A3G binding is highlighted by dashed circles and with details illustrated in insets
PDBs used for the superpositions: A3FCTD alone: 3WUS; Vif/CBFβ: 4N9F; Vif/CBFβ/A3FCTD: 6NIL; VCBC/A3G: 8CX1
The individual Vif/CBFβ structures are shown in gray
the A3F or A3G bound Vif/CBFβ are shown in magenta/cyan
Right: structural model of the HIV-1 Vif recruitment of PPP2R5C-containing PP2A onto the Cul5 E3 ligase complex
The in vitro binding assay was performed using MBP-tagged Vif/CBFβ/EloB/EloC variants to pull down SUMO-PPP2R5A variants
The SUMO-PPP2R5A bands were recognized by anti-SUMO antibody
and the His-CBFβ bands were detected by Anti-His antibody (upper panel)
The ratio of band intensities (SUMO-PPP2R5A/His-CBFβ) was quantified by mean ± sem; n = 2 biologically independent samples for R127E:WT
n = 3 biologically independent samples for WT:Y294A
n = 4 biologically independent samples for K22E:WT
n = 7 biologically independent samples for WT:WT
with individual data points shown as dots (lower panel)
<a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41594-024-01314-6#MOESM8">Source data</a>
The observed (upper panel) and computational predicted (lower panel) structures are shown in three different views to demonstrate the differences between the observed and predicted binding modes of PPP2R5A
Federation of European Biochemical Societies
An example of the raw image of the complex (left) and the particle orientation distribution of the untilted dataset (right)
Top 2D class averages of the combined untilted and tilted datasets
From a total of 516,465 particles in the classes showing preferred orientations (boxed in red)
456,465 particles (~88%) were removed and excluded randomly from the subsequent analysis
The final particle set showed a more balanced orientation distribution
The Fourier shell correlation (FSC) curves of the cryo-EM reconstruction
Local resolution estimate of the cryo-EM map
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DOI: https://doi.org/10.1038/s41594-024-01314-6
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a VIF of three or below is not a cause for concern
the less reliable your regression results are going to be
A VIF equal to one means variables are not correlated and multicollinearity does not exist in the regression model
VIF measures the strength of the correlation between the independent variables in regression analysis
This correlation is known as multicollinearity
which can cause problems for regression models
CFI. &#34;<a href="https://corporatefinanceinstitute.com/resources/knowledge/other/variance-inflation-factor-vif/" data-component="link" data-source="inlineLink" data-type="externalLink" data-ordinal="1">Variance Inflation Factor</a>.&#34;
Isixsigma. &#34;<a href="https://www.isixsigma.com/dictionary/variance-inflation-factor-vif/" data-component="link" data-source="inlineLink" data-type="externalLink" data-ordinal="1">Variance Inflation Factor (VIF)</a>.&#34;
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By <a href="https://realassets.ipe.com/ipe-staff/3274.bio" rel="author">IPE staff</a>2024-12-04T11:08:00+00:00
Vauban Infra Fibre (VIF) has&nbsp;acquired&nbsp;stakes in broadband operators in low-density areas of France from its co-shareholder Caisse des Dépôts et Consignations (CDC)
The French digital infrastructure platform has&nbsp;invested&nbsp;an undisclosed to acquire Project Chrysalide
which covers 19 fibre-to-the-home (FttH) networks and will be phased over 2024 and 2025
In September, <a href="https://realassets.ipe.com/news/gic-snaps-up-minority-interest-in-vauban-infra-fibre/10075703.article" target="_blank">Singapore’s sovereign wealth fund GIC acquired a minority stake</a> in VIF through a capital increase alongside its existing shareholders
which include funds managed by Vauban Infrastructure Partners and Crédit Agricole Assurances
Vauban-managed investment funds remain the majority stakeholders in VIF
VIF said GIC’s investment provides&nbsp;additional capital to fund future digital infrastructure investments and support long-term growth
VIF said it also expects to announce the closing of a new digital infrastructure transaction that will strengthen its position in the French market and diversify its digital infrastructure base
founding partners of Vauban Infrastructure Partners,&nbsp;said:&nbsp;“Since 2009
we have significantly expanded our digital investments by building on the recognised&nbsp;expertise of our teams and our close relationships with all the players in the sector
which&nbsp;consolidates VIF’s position as the first independent FttH platform in France
is fully in line with Vauban’s&nbsp;strategic commitment to continue its involvement as a long-term partner with all stakeholders to develop&nbsp;critical infrastructure within the territories.”
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and Fernando Galves Gen AI Solutions Architect
<a href="https://aws.amazon.com/outposts/">AWS Outposts</a> is a fully managed service that extends AWS infrastructure, services, APIs, and tools to customer premises. Outposts servers launched in 2022, a 1U or 2U rack-mountable host, with the ability to run Amazon Elastic Compute Cloud (Amazon EC2) and Amazon Elastic Container Service (Amazon ECS), as well as other appropriate smaller scale edge services such as <a href="https://aws.amazon.com/greengrass/">AWS IoT Greengrass</a>
This version of Outposts is primarily focused on bringing lower latency
AWS compute capabilities to the edge at many user locations
During Outposts provisioning, you or AWS creates a <a href="https://docs.aws.amazon.com/outposts/latest/server-userguide/service-links.html">service link connection</a> that connects your Outposts server to your chosen AWS Region or home Region
Outposts depends on regional connectivity “to reach out to home,” needing very little in terms of networking
and providing authentication details through a command line
the Outpost servers reach out to the regional endpoint
Your Outpost status will show as Active when the process has completed
it could take a few hours depending on service link bandwidth
Although this has been suitable for the vast majority of use cases
there are some locations that can’t provide internet connectivity in their environments
This has mostly been in use cases where there is a strong security reason for not having an internet connection (such as financial services kiosks
so as to avoid risks such as DDoS attacks and potential hack attempts
or to meet requirements for receiving an authority to operate (ATO)
These locations either have some form of direct connect
or more commonly have a centralized direct connect link to AWS
and an MPLS network linking all their remote sites to a central one
the requirement is to allow the Outpost servers to resolve and reach the public endpoint for setup
and subsequently the public anchor endpoint for management
This is done without needing to leave the AWS ecosystem
without needing to expose themselves unnecessarily to potential internet threats
and without adding more systems to manage themselves
we identified several key things that need to be provided if the user does not have internet connectivity at the remote location
There are three different types of <a href="https://docs.aws.amazon.com/directconnect/latest/UserGuide/create-vif.html">Virtual Interfaces (VIF)</a> possible to configure on an <a href="https://aws.amazon.com/directconnect/">AWS Direct Connect</a> link:
A transit VIF can be used to solve both of these issues
a transit VIF deploys an ENI within a VPC (known as an attachment)
so that traffic coming from the transit VIF into a VPC can be routed
the traffic has to either be sourced or targeted for an ENI in the VPC
instead of the transit gateway routing multiple VPCs to the internet
Using a transit gateway to forward traffic to an NAT gateway allows you to provide internet connectivity for the Outposts servers without managing virtual appliances
because NAT gateway provides this as a service
NAT gateways also only allow outbound access
so they provide security against any attempted external access by a bad actor from the internet
This works for Outposts servers since they only need outbound access
Outposts always initiate communication to an anchor or service endpoint
and they never receive communication except as a response
<a href="https://d2908q01vomqb2.cloudfront.net/1b6453892473a467d07372d45eb05abc2031647a/2025/03/29/image-1-9.png"></a>
Architectural diagram showing the use of a Transit VIF and NAT gateway in a Region reaching regional endpoints
Although the preceding architecture solves the challenge of how we provide a path for IP packets to transit between the Outposts servers and the public endpoints needed
it doesn’t solve the issue of resolving DNS names
If the remote site is isolated from the internet
<a href="https://docs.aws.amazon.com/Route53/latest/DeveloperGuide/outpost-resolver.html">Amazon Route53</a> resolver endpoints allow you to deploy an IP address within a VPC subnet
There are two types of resolver endpoints: outbound and inbound
Outbound resolver endpoints are used by AWS to send DNS queries to your on-premises DNS servers
Inbound resolver endpoints are used by your DNS servers (and hosts) to resolve addresses within Route 53
Route 53 can resolve public DNS names, so the Outposts service endpoint <a href="http://outposts.eu-central-1.amazonaws.com">outposts.&lt;region-name&gt;.amazonaws.com</a> becomes resolvable by an inbound resolver endpoint
Using a public VIF allows you to provide an internet connection directly to the on-premises site
this means you need to implement firewalls and security functions on this connection
adding more layers of operational overhead
A public VIF also means that the on-premises end of the VIF can be accessed by any public IP on the AWS public network
regardless of the instance to which IP is mapped
A public VIF is a public IP endpoint on the AWS public network
You should treat public VIF traffic as internet-based traffic
This can become cumbersome for firewalls teams if they have to allow-list known AWS IP ranges and manage the stateful firewall for a long range of AWS IPs
even if the user is happy to implement and manage a firewall on the end of that public VIF
there is still a question of how the Outpost would resolve DNS in this setup
Unless the private network already has DNS resolution to a public DNS
then there are no DNS servers that DHCP can point to in order to allow the Outposts servers to get name resolution
This is because there is no public DNS endpoint within the AWS public network
Traffic from a user’s public VIF can access the AWS public network
but it can’t exit it to other public networks
if the you had configured DHCP to point to one of the well-known DNS servers (such as 8.8.8.8)
since this DNS servers lives outside of the AWS public network
requests originating from the on-premises side of a public VIF would be dropped as it hit the border of the AWS autonomous system
The only way for a DNS request to be resolved would be to build a bind forwarding service within a VPC
and point the DHCP DNS values at this IP address
This network configuration introduces complexity
and won’t be possible for those with highly regulated workloads
You would need to manage a firewall on-premises
allow a public network to reach the on-premises location
and manage a bind servers setup within a VPC
a public VIF is generally not an option unless the user is already running one
and is familiar with the steps to secure it
<a href="https://d2908q01vomqb2.cloudfront.net/1b6453892473a467d07372d45eb05abc2031647a/2025/03/29/image-2-5.png"></a>
Architectural diagram showing traffic flow using a public VIF and AWS Outposts
Virtual private gateways do not have an ENI associated with them
but are pointed to as a next hop within a subnet routing table
If we take this example and look at what the Outposts servers would be trying to pass as traffic
then it would send a packet with a source address of the Outposts servers
and a destination address of the Outposts service public endpoint (assuming that it could resolve it)
then neither the source nor destination address would belong to an ENI within the VPC
Even if there was a routing rule on the subnet pointing the next hop for all traffic to a <a href="https://docs.aws.amazon.com/vpc/latest/userguide/vpc-nat-gateway.html">NAT gateway</a> (ideal for internet egress)
This is because the packet from the Outposts servers doesn’t have a destination of the NAT gateway
but instead a destination of the setup endpoint in the internet
It’s possible to use a combination of ingress routing and transparent proxies to ingest the traffic and pass it to an instance running a proxy service to forward to the internet
this adds complexity having to manage and maintain proxy servers
a private VIF is generally not recommended
<a href="https://d2908q01vomqb2.cloudfront.net/1b6453892473a467d07372d45eb05abc2031647a/2025/03/29/image-3-4.png"></a>
Architectural diagram showing VGW and packet drops because of transitive routing not being supported
In this post, we discussed architecture patterns you can use to provision your Outposts when public internet connectivity is unavailable. To get started with Outpost servers please visit our <a href="https://docs.aws.amazon.com/outposts/latest/server-userguide/what-is-outposts.html">Server User Guide</a>. For more information, <a href="https://aws.amazon.com/government-education/contact/">contact us</a> to learn more
The human immune deficiency virus (HIV) first entered public consciousness in the early 1980s
after cases of unfamiliar and deadly illnesses began to overwhelm medical centres across North America
&#8220;An estimated 42 million people have died from HIV/AIDS to date
and while people can now live full lives with access to treatments
it is still a chronic condition that people would like a cure for,&#8221; said Dr
Immunology and Biochemistry in the College of Medicine at the University of Saskatchewan (USask).&#160;&#160;
Chelico&#8217;s lab studies immune responses to HIV
particularly a family of proteins called APOBEC3 which cause mutations in the HIV virus
Her research and others&#8217; show that the virus uses a counterattack that breaks down APOBEC3&#8217;s defences
helping HIV win and establish an infection.&#160;
&#8220;There is a battle going on in the cell,&#8221; said Chelico
&#8220;HIV also has a protein known as Vif that it uses against APOBEC3
so it&#8217;s this fight against each other.&#8221;&#160;
have tried to create drugs that block Vif from interacting with APOBEC3
giving proteins the added &#8220;boost&#8221; it needs to fully inactivate the HIV virus and prevent an infection
but these blockers have been mostly ineffective.&#160;
Now, four decades after HIV was first identified, Chelico and her colleagues, including USask post-doctoral fellow Dr. Amit Gaba (PhD), have discovered previously unknown interactions between APOBEC3 and Vif proteins which offer a better map for drug design. Their findings were recently published in the American Microbiology Association&#8217;s <a href="https://doi.org/10.1128/jvi.01606-24">Journal of Virology</a>.&#160;
previous inhibitors weren&#8217;t successful because we weren&#8217;t looking at the right interface between the protein interactions,&#8221; said Chelico
&#8220;We think that with a better map of this interface we can design a better inhibitor.&#8221;&#160;
Previous studies used one Vif protein and a single APOBEC3 protein to simulate conditions during an infection
but this experimental design isn&#8217;t quite measuring up to what is actually happening inside our cells
Chelico and her team found that there are differences in the Vif proteins among HIV strains and that APOBEC3s are not working individually against HIV.&#160;
&#8220;My lab established that APOBEC3s are working together during an infection
and they form a new structure,&#8221; said Chelico
&#8220;APOBEC proteins are part of a natural defence system against viruses and if we can stop that interaction with Vif
They could be a kind of natural cure.&#8221;&#160;
this groundbreaking discovery could pave the way for new treatments
The potential for APOBEC proteins to serve as a natural defence against HIV brings a new wave of optimism in the fight against the virus.&#160;
&#8220;HIV still remains a global public health issue,&#8221; said Chelico
&#8220;Current treatments only suppress the virus so we&#8217;re really hopeful that our research offers a new avenue
where we can help the body&#8217;s natural defences
stopping HIV infections from taking hold.&#8221;&#160;
Want to learn more about Chelico&#8217;s revolutionizing work? <a href="https://www.youtube.com/watch?v=tZArE_pmb0s">Check out her 2024 TEDxUniversityofSaskatchewan talk.</a>&#160;&#160;
Together, we are addressing the world's greatest challenges. <a class="text-white" href="https://give.usask.ca">Join our ambitious vision</a> for the future
The University of Saskatchewan's main campus is situated on <a href="https://indigenous.usask.ca/indigenization-reconciliation/Land-Acknowledgement.php">Treaty 6 Territory and the Homeland of the Métis.</a>
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This article is part of the Research TopicRetroviruses, retroelements and their restrictions<a class="Link Link--linkType Link--maincolor Link--medium Link--icon Link--chevronRight Link--right" aria-label="View all 13 articles" href="https://www.frontiersin.org/research-topics/483/retroviruses-retroelements-and-their-restrictions/magazine" target="_self" data-event="customLink-linkType-a_viewAll13Articles">View all 13 articles</a>
The research on virion infectivity factor (Vif) protein had started in late 1980s right after HIV-1 was cloned
and the function of Vif had been a mystery for a long time
the research on Vif has finally lead to the identification of APOBEC3G
which opens up a new era in the research field of host restriction factors in HIV-1 infection followed by TRIM5&#x003B1;
This suggests that continuation of basic research on fundamental questions is quite important
We still have many questions on Vif and APOBEC3 and should continue to work on these proteins in the future in order to better regulate HIV-1
We will discuss not only the history but also recent advances in Vif research
The underlying mechanism of Vif function had been unsolved and a mystery for a long time
In addition to the above described main function, early studies also revealed several important Vif functions including dimerization (<a href="#B54">Yang et al., 2001</a>), virion incorporation (<a href="#B3">Camaur and Trono, 1996</a>; <a href="#B45">Simon et al., 1997</a>), and phosphorylation (<a href="#B56">Yang et al., 1996</a>; <a href="#B55">Yang and Gabuzda, 1998</a>); however
the significances of these functions are not discussed much recently
a novel Vif function on cell cycle has been reported
Schematic figure of the virion infectivity factor (Vif) protein and amino acid motifs for binding to Vif-interacting proteins
Pink indicates binding motifs for A3G; light blue indicates binding motifs for A3F; light green indicates binding motifs for Cul5; yellow indicates binding motifs for EloC; light pink indicates motifs for dimerization
Vif binds to p53 and CBF&#x003B2; in its N-terminal regions
but binding motifs were not elucidated yet
It is quite important to reveal the interaction sites between Vif and APOBEC3 proteins
because the regulation of this interaction may lead to the development of novel therapeutic strategies for HIV-1 infection
their structural information is not fully elucidated yet
because it is quite difficult to produce these proteins as soluble forms
the information described below is mainly obtained by many studies using site-directed mutagenesis
we have to wait a little longer until we will get the structural information of these complexes
the mechanisms by which CBF&#x003B2; regulates the E3 ligase complex are still under investigation
since CBF&#x003B2; is an important T cell transcription factor
it would be very interesting to determine whether Vif affects T cell differentiation
The identification of the E3 ligase has lead to elucidation of the mechanisms of Vif-induced G2 cell cycle arrest described below
The mechanisms how Vif is ubiquitinated and degraded and how Vif induces G2 cell cycle arrest
Vif inhibits ubiquitination of p53 by MDM2 to induce activation and nuclear import of p53
Activated p53 induces transcription of several genes including MDM2 and p21
Enhanced expression of MDM2 may lead to more Vif ubiquitination and degradation
which forms the autoregulatory circuit of Vif expression
activation of p21 leads to G2 cell cycle arrest
HIV-1 needs to have G2 cell cycle arrest to efficiently replicate so that it possesses two accessory genes such as vif and vpr
Vif induces G2 arrest in a p53-dependent manner
while Vpr accomplishes the same goal in a p53-independent manner
not only because it opens up a new era in the research field of host restriction factors
but also because it has a variety of functions for the viral life cycle by interacting several cellular proteins
It suggests that it might be a good target for control of HIV-1 infection
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest
This work was partly supported by grants-in-aid from the Ministry of Education
and Technology and from the Ministry of Health
This work was also partly supported by grants from SENSHIN Medical Research Foundation
High level expression of human immunodeficiency virus type-1 Vif inhibits viral infectivity by modulating proteolytic processing of the Gag precursor at the p2/nucleocapsid processing site
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=14722068" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=14722068" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1074/jbc.M312426200" target="_blank">CrossRef Full Text</a>
A single amino acid difference in the host APOBEC3G protein controls the primate species specificity of HIV type 1 virion infectivity factor
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=14999100" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=14999100" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1073/pnas.0307713101" target="_blank">CrossRef Full Text</a>
Characterization of human immunodeficiency virus type 1 Vif particle incorporation
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=8709234" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=8709234" target="_blank">Pubmed Full Text</a>
A patch of positively charged amino acids surrounding the human immunodeficiency virus type 1 Vif SLVx4Yx9Y motif influences its interaction with APOBEC3G
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Identification of a novel WxSLVK motif in the N terminus of human immunodeficiency virus and simian immunodeficiency virus Vif that is critical for APOBEC3G and APOBEC3F neutralization
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Identification of highly attenuated mutants of simian immunodeficiency virus
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The Vif protein of human immunodeficiency virus type 1 is posttranslationally modified by ubiquitin
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=15013426" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=15013426" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1016/j.bbrc.2004.01.023" target="_blank">CrossRef Full Text</a>
The sor gene of HIV-1 is required for efficient virus transmission in vitro
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=3497453" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=3497453" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1126/science.3497453" target="_blank">CrossRef Full Text</a>
Expression of HIV-1 accessory protein Vif is controlled uniquely to be low and optimal by proteasome degradation
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=15374000" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=15374000" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1016/j.micinf.2004.04.011" target="_blank">CrossRef Full Text</a>
Role of vif in replication of human immunodeficiency virus type 1 in CD4+ T lymphocytes
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Insights into the dual activity of SIVmac239 Vif against human and African green monkey APOBEC3G
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=23189135" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=23189135" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1371/journal.pone.0048850" target="_blank">CrossRef Full Text</a>
HIV-1 Vpr increases viral expression by manipulation of the cell cycle: a mechanism for selection of Vpr in vivo
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<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=18577210" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=18577210" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1186/1742-4690-5-51" target="_blank">CrossRef Full Text</a>
Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=7474080" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=7474080" target="_blank">Pubmed Full Text</a>
Characterization of conserved motifs in HIV-1 Vif required for APOBEC3G and APOBEC3F interaction
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=18619467" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=18619467" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1016/j.jmb.2008.06.061" target="_blank">CrossRef Full Text</a>
Identification of amino acid residues in APOBEC3G required for regulation by human immunodeficiency virus type 1 Vif and Virion encapsidation
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=17267497" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=17267497" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1128/JVI.02795-06" target="_blank">CrossRef Full Text</a>
HIV-1 Vif-mediated ubiquitination/degradation of APOBEC3G involves four critical lysine residues in its C-terminal domain
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Structural features of antiviral APOBEC3 proteins are linked to their functional activities
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=22203821" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=22203821" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.3389/fmicb.2011.00258" target="_blank">CrossRef Full Text</a>
Ubiquitination of APOBEC3G by an HIV-1 Vif&#x02013;Cullin5&#x02013;Elongin B&#x02013;Elongin C complex is essential for Vif function
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Primate lentiviral virion infectivity factors are substrate receptors that assemble with cullin 5-E3 ligase through a HCCH motif to suppress APOBEC3G
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=16076960" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=16076960" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1073/pnas.0502440102" target="_blank">CrossRef Full Text</a>
An endogenous inhibitor of human immunodeficiency virus in human lymphocytes is overcome by the viral Vif protein
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=9811770" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=9811770" target="_blank">Pubmed Full Text</a>
A single amino acid determinant governs the species-specific sensitivity of APOBEC3G to Vif action
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=14966139" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=14966139" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1074/jbc.C400060200" target="_blank">CrossRef Full Text</a>
Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=12859895" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=12859895" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1016/S0092-8674(03)00515-4" target="_blank">CrossRef Full Text</a>
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Phosphorylation of a novel SOCS-box regulates assembly of the HIV-1 Vif&#x02013;Cul5 complex that promotes APOBEC3G degradation
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Vif Overcomes the innate antiviral activity of APOBEC3G by promoting its degradation in the ubiquitin&#x02013;proteasome pathway
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A zinc-binding region in Vif binds Cul5 and determines cullin selection
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HIV-1 Vpr induces ATM-dependent cellular signal with enhanced homologous recombination
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Human immunodeficiency virus type 1 Vif inhibits packaging and antiviral activity of a degradation-resistant APOBEC3G variant
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Regulation of APOBEC3 proteins by a novel YXXL motif in human immunodeficiency virus type 1 Vif and simian immunodeficiency virus SIVagm Vif
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Human immunodeficiency virus type 1 Vpr arrests the cell cycle in G2 by inhibiting the activation of p34cdc2-cyclin B
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Activation of the ATR-mediated DNA Damage Response by the HIV-1 viral protein R
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=12738771" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=12738771" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1074/jbc.M303948200" target="_blank">CrossRef Full Text</a>
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Cell-dependent requirement of human immunodeficiency virus type 1 Vif protein for maturation of virus particles
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A single amino acid of APOBEC3G controls its species-specific interaction with virion infectivity factor (Vif)
<a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&#x00026;Cmd=ShowDetailView&#x00026;TermToSearch=14978281" target="_blank">Pubmed Abstract</a> | <a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?db=pubmed&#x00026;cmd=prlinks&#x00026;retmode=ref&#x00026;id=14978281" target="_blank">Pubmed Full Text</a> | <a href="http://dx.doi.org/10.1073/pnas.0307132101" target="_blank">CrossRef Full Text</a>
Polyubiquitination of APOBEC3G is essential for its degradation by HIV-1 Vif
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Ubiquitination of APOBEC3 proteins by the Vif&#x02013;Cullin5&#x02013;ElonginB&#x02013;ElonginC complex
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Phosphorylation of APOBEC3G by protein kinase A regulates its interaction with HIV-1 Vif
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The Vif and Gag proteins of human immunodeficiency virus type 1 colocalize in infected human T cells
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Evidence for a newly discovered cellular anti-HIV-1 phenotype
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Citation: Takaori-Kondo A and Shindo K (2013) HIV-1 Vif: a guardian of the virus that opens up a new era in the research field of restriction factors. Front. Microbio. 4:34. <a href="http://dx.doi.org/10.3389/fmicb.2013.00034">doi: 10.3389/fmicb.2013.00034</a>
Copyright: &#x000A9; 2013 Takaori-Kondo and Shindo. This is an open-access article distributed under the terms of the <a id="lnkNonCommercialLicense" href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</a>
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provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc
*Correspondence: Akifumi Takaori-Kondo, Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Shogoin-Kawaracho 54, Sakyo-ku, Kyoto 606-8507, Japan. e-mail:<a id="encmail">YXRha2FvcmlAa3VocC5reW90by11LmFjLmpw</a>
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that antagonizes A3 family members by targeting them for degradation
Diversification of A3 allows host escape from Vif whereas adaptations in Vif enable cross-species transmission of primate lentiviruses
How this ‘molecular arms race’ plays out at the structural level is unknown
we report the cryogenic electron microscopy structure of human APOBEC3G (A3G) bound to HIV-1 Vif
and the hijacked cellular proteins that promote ubiquitin-mediated proteolysis
A small surface explains the molecular arms race
including a cross-species transmission event that led to the birth of HIV-1
we find that RNA is a molecular glue for the Vif–A3G interaction
enabling Vif to repress A3G by ubiquitin-dependent and -independent mechanisms
Our results suggest a model in which Vif antagonizes A3G by intercepting it in its most dangerous form for the virus—when bound to RNA and on the pathway to packaging—to prevent viral restriction
By engaging essential surfaces required for restriction
suggesting a general mechanism by which RNA binding helps to position key residues necessary for viral antagonism of a host antiviral gene
Although it is commonly assumed that sites of molecular arms races report on direct protein interactions
physical evidence of this interaction site to explain the mechanisms of how Vif promotes processive ubiquitination on A3G
and how mutations in Vif or A3G promote host escape and viral adaptation
leaving substantial gaps in our knowledge of molecular mechanisms of Vif antagonism of A3 proteins and molecular arms races between them
logo plot of amino acids found in the consensus of all HIV-1 clades
as well as SIVcpz (black bar) and all other SIV strains with equal distribution of each SIV (white bar)
Both this result and our structure suggest that A3G bound to purine-rich RNA is the substrate of the Vif E3 ligase
which validates their importance in our structure
Ribbon diagram showing position of molecular arms race interface (spheres) relative to the RNA interface (sticks)
Close-up of molecular arms race interface (top) and residues that contribute to Vif–A3G binding and in contact with RNA (bottom)
Residues D128 and D130 of A3G are under diversifying selection; residue Q83 is an adaptation that allowed SIVrcm Vif to neutralize hominid primate A3G and thus enable cross-species transmission
Logo plots of natural sequence variation in Vif residues that line the molecular arms race (top) and Vif–A3G–RNA interface (bottom)
HIV-1 and SIVcpz sequences (black bars) are the consensus of all HIV-1 clades as well as SIVcpz
and SIV sequences (white bars) are all other SIV strains using equal distribution of each SIV
We suggest that structural plasticity in Vif enabled amino acid substitutions
to neutralize A3G and enable cross-species transmission of SIV from red-capped mangabeys to chimpanzees
We conclude that Vif binds A3G/RNA in a manner that limits A3G escape over long evolutionary timescales by engaging an essential surface required for antiviral function
explaining why genetic signatures of diversifying selection and adaptation are constrained to the direct protein interactions observed at the molecular arms race interface
Bottom panels show close-up of interactions within A3G CDA domains
This observation indicates that VCBC binds A3G in a manner that inhibits its self-association
We suggest that these surfaces may be bridged by cellular cofactors as described for A3G
possibility is that interactions with Vif are stabilized by bipartite interactions with tandem CDA domains of A3 proteins
Structural studies of Vif–A3 complexes purified after coexpression or native purification from eukaryotic cells will allow this question to be addressed in future studies
We propose that the substrate of the Vif E3 ligase is not A3G but rather a complex of A3G bound to purine-rich RNA
including purine-rich sequences found in the viral genome
This finding suggests that Vif binding to A3G has the capacity to block its packaging independent of ubiquitination activity
a mechanism that may potentiate repression of restriction
</a>a,b, Packaging of A3G into HIV-1 virus requires A3G dimerization and its interaction with viral RNA (a); Vif neutralizes A3G early during its biosynthesis by binding RNA-bound A3G, inhibition of A3G dimerization and promotion of ubiquitin-mediated proteolysis (b). Created with <a href="http://BioRender.com">BioRender.com</a>
A defocus range of −0.8 to −2.0 μm was applied
The weak density in this region precluded precise atomic modelling
and thus the A3G–Vif dimeric interface for state 2 is interpretable on one side only
We generated a library of variants at positions 22
26 and 40 using degenerate oligonucleotide mutagenesis in the HIV-1 LAI vif gene
Individual colonies were sequenced and ligated into a lentiviral vector
flanked by a C-terminal 3XFLAG epitope tag in the pcDNA4/TO vector backbone (Thermo Fisher
was transfected into HEK293T cells (ATCC CRL-3216
regularly tested for mycoplasma contamination) plated in six-well dishes at a density of 1.5 × 105 cells ml–1
The amount of A3G packaged into virions was assayed by cotransfection of 1,000 ng of Vif vector
200 ng of A3G-3XFlag and 500 ng of psPAX2 for gag/pol production with TransIT-LT1 transfection reagent (Mirus
MIR2304) at a reagent to plasmid DNA ratio of 3:1
1 ml of the supernatant was filtered through a 0.2 μm syringe filter and virions were pelleted in an Eppindorf 5415R tabletop microcentrifuge for 1 h at 4 °C and maximum speed
and 25 μl of NuPAGE 4× loading dye (Invitrogen no
Samples were boiled for 10 min at 95 °C and loaded on an SDS–PAGE gel
3537) antibodies were used for immunoblotting at a dilution of 1:5,000
Mouse IgG HRP-conjugated antibody (R&amp;D systems
HAF007) was used to detect primary antibodies at a dilution of 1:5,000
Chemiluminescent signals from all immunoblots were imaged using the ChemiDocMP imaging system (Bio-Rad)
and images were processed with ImageJ software to quantify the densitometry for each detected antibody band
Normalized A3G in virions was calculated by dividing the amount of A3G by that of p24gag and setting that number to 1.0 for the ‘No Vif’ control
Further information on research design is available in the <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41586-023-05779-1#MOESM2">Nature Portfolio Reporting Summary</a> linked to this article
Structural insights into APOBEC3-mediated lentiviral restriction
The battle between retroviruses and APOBEC3 genes: its past and present
HIV-1 uncoats in the nucleus near sites of integration
Reconstitution and visualization of HIV-1 capsid-dependent replication and integration in vitro
Nuclear pore blockade reveals that HIV-1 completes reverse transcription and uncoating in the nucleus
RNA-dependent oligomerization of APOBEC3G is required for restriction of HIV-1
Identification of amino acid residues in APOBEC3G required for regulation by human immunodeficiency virus type 1 Vif and virion encapsidation
Transcriptional regulation of APOBEC3 antiviral immunity through the CBF-beta/RUNX axis
Fab-based inhibitors reveal ubiquitin independent functions for HIV Vif neutralization of APOBEC3 restriction factors
Human immunodeficiency virus type 1 Vif protein is packaged into the nucleoprotein complex through an interaction with viral genomic RNA
Ancient adaptive evolution of the primate antiviral DNA-editing enzyme APOBEC3G
The host restriction factor APOBEC3G and retroviral Vif protein coevolve due to ongoing genetic conflict
Rules of engagement: molecular insights from host-virus arms races
Gene loss and adaptation to hominids underlie the ancient origin of HIV-1
Structure of the Vif-binding domain of the antiviral enzyme APOBEC3G
Crystal structure of a soluble APOBEC3G variant suggests ssDNA to bind in a channel that extends between the two domains
Understanding the structural basis of HIV-1 restriction by the full length double-domain APOBEC3G
Structural determinants of HIV-1 Vif susceptibility and DNA binding in APOBEC3F
Conformational dynamics of the HIV-Vif protein complex
Host gene evolution traces the evolutionary history of ancient primate lentiviruses
Function analysis of sequences in human APOBEC3G involved in Vif-mediated degradation
The Vif protein of HIV triggers degradation of the human antiretroviral DNA deaminase APOBEC3G
Functional analysis and structural modeling of human APOBEC3G reveal the role of evolutionarily conserved elements in the inhibition of human immunodeficiency virus type 1 infection and Alu transposition
Antiviral protein APOBEC3G localizes to ribonucleoprotein complexes found in P bodies and stress granules
Two regions within the amino-terminal half of APOBEC3G cooperate to determine cytoplasmic localization
The anti-HIV-1 editing enzyme APOBEC3G binds HIV-1 RNA and messenger RNAs that shuttle between polysomes and stress granules
Identification of amino acid residues in HIV-1 Vif critical for binding and exclusion of APOBEC3G/F
Vif proteins from diverse primate lentiviral lineages use the same binding site in APOBEC3G
The RNA binding specificity of human APOBEC3 proteins resembles that of HIV-1 nucleocapsid
Mechanism of auxin perception by the TIR1 ubiquitin ligase
Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide
Examination of the APOBEC3 barrier to cross species transmission of primate lentiviruses
Structural basis for a species-specific determinant of an SIV Vif protein toward hominid APOBEC3G antagonism
Ubiquitin ligation to F-box protein targets by SCF-RBR E3-E3 super-assembly
Dispersed sites of HIV Vif-dependent polyubiquitination in the DNA deaminase APOBEC3F
ARIH2 is a Vif-dependent regulator of CUL5-mediated APOBEC3G degradation in HIV infection
The biased nucleotide composition of the HIV genome: a constant factor in a highly variable virus
Newly synthesized APOBEC3G is incorporated into HIV virions
Differential sensitivity of “old” versus “new” APOBEC3G to human immunodeficiency virus type 1 vif
MacroBac: new technologies for robust and efficient large-scale production of recombinant multiprotein complexes
SerialEM: a program for automated tilt series acquisition on Tecnai microscopes using prediction of specimen position
Scipion: a software framework toward integration
reproducibility and validation in 3D electron microscopy
MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy
Sampling the conformational space of the catalytic subunit of human gamma-secretase
Visualizing density maps with UCSF Chimera
RELION: implementation of a Bayesian approach to cryo-EM structure determination
3D variability analysis: resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM
Optimal determination of particle orientation
and contrast loss in single-particle electron cryomicroscopy
Improvement of cryo-EM maps by density modification
Quantifying the local resolution of cryo-EM density maps
Addressing preferred specimen orientation in single-particle cryo-EM through tilting
Asarnow, D., Palovcak, E. &amp; Cheng, Y. asarnow/pyem:UCSF pyem v0.5. Zenodo <a href="https://zenodo.org/record/3576630#.Y-Tme3bP3IU" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="https://zenodo.org/record/3576630#.Y-Tme3bP3IU">https://zenodo.org/record/3576630#.Y-Tme3bP3IU</a> (2019)
Comparative protein modelling by satisfaction of spatial restraints
Comparative protein structure modeling using MODELLER
Coot: model-building tools for molecular graphics
ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps
Real-space refinement in PHENIX for cryo-EM and crystallography
Haruspex: a neural network for the automatic identification of oligonucleotides and protein secondary structure in cryo-electron microscopy maps
Correcting pervasive errors in RNA crystallography through enumerative structure prediction
MolProbity: all-atom structure validation for macromolecular crystallography
Measurement of atom resolvability in cryo-EM maps with Q-scores
BINANA 2: characterizing receptor/ligand interactions in Python and JavaScript
PLIP: fully automated protein-ligand interaction profiler
LigPlot+: multiple ligand-protein interaction diagrams for drug discovery
Schrödinger, L. &amp; DeLano, W. L. PyMOL. <a href="http://www.pymol.org/pymol" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="http://www.pymol.org/pymol">http://www.pymol.org/pymol</a> (2020)
UCSF ChimeraX: meeting modern challenges in visualization and analysis
Statistical potential for assessment and prediction of protein structures
CUL5-ARIH2 E3-E3 ubiquitin ligase structure reveals cullin-specific NEDD8 activation
Deciphering key features in protein structures with the new ENDscript server
<a data-track="click" data-track-action="download citation references" data-track-label="link" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/s41586-023-05779-1?format=refman&amp;flavour=references">Download references</a>
Cheng and Gross laboratories for helpful discussions on this project; D
Bulkley of UCSF Cryo-EM facility for assistance with data collection; S
Gradia of Macrolab for insect cell expression vectors; and A
Manglik of UCSF for advice on model building and refinement
was supported by the University of Washington STD/AIDS Research Training Fellowship (no
NIH/NIAID T32-AI07140) and by a 2021 New Investigator Award from the University of Washington/Fred Hutch Center for AIDS Research (NIH-funded programme under award no
was supported by a University of Washington Cellular and Molecular Biology Training Grant (no
This work was supported by funding from NIH P50AI150476 and U54AI170792 to M.E.
Divisions of Human Biology and Basic Sciences
Molecular and Cellular Biology Graduate Program
Department of Cellular and Molecular Pharmacology
Department of Bioengineering and Therapeutic Sciences
designed and expressed purified protein complexes
collected and processed cryo-EM data and performed model building and refinement
developed and performed functional experiments and evolutionary analysis
in microscope operation and cryo-EM data acquisition
helped with EM data analysis and interpretation
checked all published mutants of A3G and Vif
All authors contributed to data interpretation
Similar class averages were obtained from three independent preparations imaged on Quantifoil Gold grids
Shown at the bottom are expanded views of the fourth and fifth 2D classes with two copies of A3G-VCBC labeled in white and yellow
Shown on the right are the A3G and VCBC structures fit in the consensus map that is colored by subunits
Masks were used to determine different regions of volume for focused refinement and 3D variability analysis in cryoSPARC
Green and orange boxes indicate the final reconstruction for monomer and dimers
respectively; their corresponding Gold-Standard Fourier Shell Correlation (GSFSC) curves are shown at the bottom
The nominal resolution of the final map for monomer
Right of each panel: Logo plot of amino acids found in the consensus of all HIV-1 clades as well as SIVcpz (black bar)
and all other SIV strains using equal distribution of each SIV (white bar)
Residue F46 and W74 of SIVrcm Vif previously reported to be critical for rcmA3G neutralization engage in extensive hydrophobic interactions with rcmA3G in the model (bottom)
Note amino acids 16 and 86 of SIVrcm Vif correspond to amino acids 15 and 83
Sequence alignment of A3G residues that contact RNA or Vif from Old World Monkeys and hominids
Fully conserved residues are highlighted with white text on black background
Buried solvent accessible surface area for A3G–RNA–VCBC monomer structure
Supplementary discussion: this file discusses the structure of A3G-RNA-VCBC dimeric complexes
1 contains uncropped source images for Fig
2–4 show the model–map fit for A3G-RNA-VCBC monomeric and dimeric complexes
Supplementary Table 1 summarizes Vif residues reportedly involved in RNA binding based on mutational analyses
Supplementary Table 2 summarizes A3G residues reportedly involved in RNA binding based on mutational analyses
Comparison of dimeric complex structure in states 1 and 2
States 1 and 2 are related by rigid-body motion of A3G-RNA-VCBC protomers
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Human APOBEC3 (A3) cytidine deaminases are antiviral factors that are particularly potent against retroviruses
HIV-1 uses a viral infectivity factor (Vif) to target specific human A3s for proteasomal degradation
Vif recruits cellular transcription cofactor CBF-β and Cullin-5 (CUL5) RING E3 ubiquitin ligase to bind different A3s distinctively
but how this is accomplished remains unclear in the absence of the atomic structure of the complex
we present the cryo-EM structures of HIV-1 Vif in complex with human A3H
CBF-β and components of CUL5 ubiquitin ligase (CUL5
Vif nucleates the entire complex by directly binding four human proteins
The structures reveal a large interface area between A3H and Vif
primarily mediated by an α-helical side of A3H and a five-stranded β-sheet of Vif
This A3H-Vif interface unveils the basis for sensitivity-modulating polymorphism of both proteins
including a previously reported gain-of-function mutation in Vif isolated from HIV/AIDS patients
Our structural and functional results provide insights into the remarkable interplay between HIV and humans and would inform development efforts for anti-HIV therapeutics
how these multiple interacting interfaces contribute to the remarkable interplay between HIV and host remains incomplete due to the lack of high-resolution structures of Vif-host factor complexes
We report here the cryo-EM structures of human A3H bound to HIV-1 Vif in complex with CBF-β and multiple components of CUL5 E3 ligase
The structures reveal the architecture of the multicomponent HIV-human protein complex and illuminate the molecular details of the crucial interface between A3H and Vif that enables stable polyubiquitin chain formation and degradation of A3H
Our study further provides insights into the A3H ubiquitination sites targeted by the Vif-CUL5 E3 ligase complex
</a>a Alignment of Vif sequences from various HIV-1/SIV strains
At amino acid position 48 (marked by a triangle)
At amino acid position 97 (marked by a triangle)
whereas chimpanzee A3H has glutamine (Q97)
Conserved zinc-coordinating cysteines are marked by asterisks
c Vif-mediated degradation assay of A3H in HEK293T cells
and steady-state levels of A3H at post-48 h transfection were analyzed by Western blots
Quantified A3H levels are shown in the right-side chart
Assays were performed independently in triplicate (mean ± s.d.; n = 3)
The statistical significance of A3H degradation was assessed by two-tailed t-test assuming equal variance; *P &lt; 0.05; **P &lt; 0.005; ***P &lt; 0.0005; NS
The addition of N48H mutation in Vif lowered the levels of both WT and K97Q A3H
K97Q mutation in A3H lowered the A3H levels when transfected with either WT or N48H Vif
An anti-α-tubulin antibody was used as a loading control
d Binding analysis of A3H and VCBCCR complex by SEC
The biggest peak shift was observed when K97Q A3H was combined with N48H NL4-3 Vif
The fractions corresponding to peak 1 were used for the cryo-EM study
SDS-PAGE gels show the protein components in the indicated SEC fractions
Vif-mediated ubiquitin chain initiation and elongation on the purified A3H were tested with or without ubiquitin-conjugating enzymes ARIH2/UBE2L3 and UBE2R1
Mono-ubiquitination and poly-ubiquitination were the most efficient when the K97Q A3H was combined with N48H NL4-3 Vif
Source data are provided as a Source Data file
In vitro ubiquitination assay showed essentially the same trend, as the addition of K97Q mutation enhanced the rate of mono- and poly-ubiquitination of A3H, and the most efficient ubiquitination pattern was observed when K97Q A3H was combined with N48H Vif (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41467-023-40955-x#Fig1">1e</a>)
these results highlight the determinants of molecular interaction between A3H and Vif and demonstrate that the resulting protein complex is functional in vitro
</a>a Domain organization and construct design of A3H and VCBCC complex
Gray lines are not included in the construct
the invisible regions in the cryo-EM map are colored in gray
The key point-mutations for the stable complex assembly are marked with stars
b 3.2 Å cryo-EM reconstruction (top) and resultant atomic model (bottom) of the A3H-VCBCC complex
and RNA are shown in semi-transparent density superposed with corresponding atomic models (sticks)
</a>a Structure of the A3H-Vif subregion in the A3H-VCBCC complex
b Close-up view of the interface between A3H α3 and Vif
c Close-up view of the interface between A3H α4 and Vif
d Interface between A3H loops 4 and 6 and Vif
e Schematic of the amino acid residues involved in the A3H-Vif interactions
g Vif-mediated degradation assay of A3H mutants
Mutations were introduced in the Vif-interface residues around α3 and α4 (f) and around loops 4 and 6 (g) on top of K97Q Vif-sensitivity enhancing mutation
The A3H levels in the presence and absence of Vif (+N48H) were probed by Western blots
h Vif-mediated degradation assay using Vif mutants
Mutations were introduced in the A3H-interface residues on top of N48H
A3H levels in the presence of Vif mutants were probed by Western blots
Quantified A3H levels are shown in the right chart
These results indicate that while the α3 and α4 of A3H are major structural components of Vif-interface
the two electrostatic interactions outside the α3-α4 patch are also essential to the A3H-Vif interaction
The results also indicate that the A3H E70–Vif R93 pair plays a more dominant role than A3H D100–Vif K92 pair
</a>a Spatial relationship of A3H and C/R module for ubiquitin transfer based on the fitted atomic models of A3H-VCBCCR complex
b Clustered surface-exposed lysine residues around loops 1 and 3 of A3H
c Sequence alignment of primate A3H highlighting the conserved lysine residues in loops 1 and 3 that contain a lysine-rich patch
Numbers above the sequence correspond to residue numbering in hA3H
d Vif-mediated degradation assay of A3H lysine mutants
Lysines in loops 1 and 3 were replaced with arginines
which would maintain the positively charged side chains but block ubiquitination
The combination of four lysine-to-arginine mutations rendered A3H largely resistant to Vif-mediated degradation
The mutations were introduced on top of the K97Q Vif-sensitivity enhancing mutation
Quantified A3H levels are shown next to the gel image
</a>a Comparison of surface areas responsible for Vif binding
and ubiquitination in A3H (left) and A3G (right)
b Vif surface residues responsible for A3H binding (sticks in cyan) and A3G-RNA binding (sticks in blue)
Y30 is the only residue involved in both complexes (stick in pink)
c Atomic model showing key amino-acid contacts modulating the A3H-Vif interaction
the glutamine forms a hydrogen bond with Vif K63
in a gain-of-function HIV strain that has adapted to A3H-mediated HIV restriction
Vif E63 can form an electrostatic interaction with K97
indicating that no major conformational change in RNA is induced upon the binding to Vif
Two complex structures enabled us to map Vif amino acid residues for both A3G-RNA and A3H binding (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41467-023-40955-x#Fig5">5b</a>)
A3G-RNA-binding residues largely reside around the α1 and loop 3
while A3H-binding residues reside around the 5-stranded β-sheets and loop 3
Y30 at the end of α1 is the only residue directly involved in both complex formations
Vif loop 3 is also central to both A3G-RNA and A3H binding
while no Vif loop 3 residues are directly involved in both A3G and A3H binding
both demonstrated that A3H residues on α3 and α4 contribute to the sensitivity to Vif in cell-based degradation assays
further generated a structural model by using anchor points revealed by the Vif adaptation experiments
they demonstrated that Vif mutation E45Q can restore the degradation of Vif-resistant A3H mutant S86E
The cryo-EM structure we obtained here largely agrees with the predicted model
we did observe a polar interaction between A3H S86 and Vif E45 in the structure
these studies showed that HIV clones with K63E in Vif resulted in dramatically elevated infectivity and enhanced replication kinetics in the presence of A3H expression
suggesting more effective inactivation of A3H antiviral activity
this gain-of-function mutation may represent a possible evolutionary route for the current HIV strains
the critical α4 residue W90 is either glycine or arginine in Old world monkey A3Hs
Notwithstanding the molecular details uncovered here for the interface between human A3H and HIV Vif
further work is desirable to unveil how Vif-mediated antagonism of A3H is accomplished in other primates and their cognate SIV strains
The atomic and chemical details uncovered in this study would be helpful for future HIV therapeutic efforts targeting this fundamental virus-host interface
FLAG-A3H-pcDNA or HA-A3H-pcDNA variants were co-transfected with either Vif-pcDNA or pcDNA3.1(+) empty vector into HEK293T cells (ATCC) by using X-tremeGENE 9 DNA Transfection Reagent (Roche)
the cells were washed once with PBS and lysed in RIPA buffer with 1× complete protease inhibitors (Roche)
The lysates were then subjected to Western blot with anti-FLAG M2 mAb from mouse (Sigma-Aldrich
1:5000) and anti-Vif mAb from mouse (NIH AIDS Reagent Program #319
Cy3-labeled goat-anti-mouse mAb (GE Healthcare
1:3000 dilution) was used as a secondary antibody to detect the signal
The fluorescent signal was detected and visualized with Typhoon RGB Biomolecular Imager (GE Healthcare)
and His6-RBX2/GB1-CUL5-pETDuet-1 expression vectors were transformed into the E
and A3H-pMAL-c5X expression vector was transformed into C43 (DE3) pLysS
coli cells harboring the expression vectors were grown in an LB medium at 37 °C until the OD600 reached 0.6
The recombinant proteins were induced by 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16 °C for 18 h
the cell pellets were resuspended with buffer A (20 mM Tris-HCl (pH 8.0)
and 0.5 mM TCEP) containing RNase A (0.1 mg/ml
and cellular debris was removed by centrifugation
The supernatant containing the His6-VCBC complex was loaded onto the Ni-NTA agarose column (Qiagen)
The nickel column was extensively washed with wash buffer (20 mM Tris-HCl (pH 8.0)
and the protein was eluted with elution buffer (20 mM Tris-HCl (pH 8.0)
The His6-tag was cleaved by incubating with PreScission protease overnight
VCBC complex was subjected to HiLoad 16/600 Superdex 200 pg column (Cytiva) equilibrated with buffer A
The peak fractions were collected and concentrated for mixing with either CUL5-NTD or CUL5/RBX2 complex
the cell pellets were resuspended with buffer A
The supernatant containing GST-CUL5-NTD was loaded onto glutathione sepharose column (GE Healthcare)
The glutathione column was extensively washed with buffer A and the GST tag was cleaved with PreScission protease on the column overnight
CUL5-NTD was eluted from the glutathione column and subjected to HiLoad 16/600 Superdex 75 pg column (GE Healthcare) equilibrated with buffer A
The peak fractions were collected and concentrated for mixing with the VCBC complex
The supernatant containing His6-RBX2/GB1-CUL5 was loaded onto the Ni-NTA agarose column
The His6-tag and GB1-tag were cleaved by incubating with PreScission protease overnight
CUL5/RBX2 complex was subjected to Superdex 200 Increase 10/300 GL column (Cytiva) equilibrated with buffer A
the cell pellets were resuspended with the buffer B (25 mM HEPES-NaOH (pH 7.5)
and 0.5 mM TCEP) containing RNase A (0.1 mg/ml)
The supernatant containing MBP-A3H was loaded onto amylose column (New England Biolabs)
The amylose column was extensively washed with wash buffer (25 mM HEPES-NaOH (pH 7.5)
and the protein was eluted with the elution buffer (25 mM HEPES-NaOH (pH 7.5)
Eluted fractions were concentrated and subjected to HiLoad 16/600 Superdex 200 pg column (Cytiva) equilibrated with buffer B
Peak fractions of dimeric MBP-A3H were separated and concentrated
VCBCC complex and VCBCCR complexes were formed by mixing VCBC complex and CUL5-NTD or CUL5/RBX2 by 1:1 molar ratio and incubating on ice for 30 min
The protein complex was purified by Superdex 200 Increase 10/300 GL column
MBP-tag was cleaved from A3H with PreScission protease
A3H-VCBCC and A3H-VCBCCR complexes were formed by mixing them at a 1:1 molar ratio and incubating on ice for 30 min
The protein mix was further purified by Superdex 200 Increase 10/300 GL column
The peak fraction was isolated and concentrated for cryo-EM work
ARIH2, UBE2L3 and UBE2R1 proteins for in vitro ubiquitination assay were purified as previously described<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 37" title="Ito, F. et al. Structural basis for HIV-1 antagonism of host APOBEC3G via Cullin E3 ligase. Sci. Adv. 9, eade3168 (2023)." href="/articles/s41467-023-40955-x#ref-CR37" id="ref-link-section-d451037910e1353">37</a>
Protein purity and stoichiometry of the protein complexes were assessed by SDS-PAGE at each purification step
Purified MBP-A3H and VCBCCR complex were mixed by 1:1 molar ratio in binding buffer (25 mM HEPES-NaOH (pH 7.5)
and 0.5 mM TCEP) and incubated at 4 °C for 30 min
The mixture was then subjected to Superdex 200 Increase 10/300 GL column equilibrated with the binding buffer
Fractions were concentrated and visualized by SDS-PAGE
NEDD8 was conjugated to CUL5 in the VCBCCR complex with either WT or N48H Vif to activate the E3 ubiquitin ligase for recruiting E2 ubiquitin-conjugating enzymes
The NEDD8 conjugation reaction was performed by mixing 10 μM VCBCCR complex
0.8 μM E1 NEDD8 activating enzyme (Enzo Life Sciences)
15 U/ml inorganic pyrophosphatase from baker’s yeast (Sigma-Aldrich)
1 mM DTT and incubating for 2.5 h at room temperature
NEDD8-conjugated VCBCCR (N8~VCBCCR) complex was purified by Superdex 200 Increase 10/300 GL column to remove excess amount of NEDD8 and other enzymes in the reaction
MBP-tag was cleaved from A3H with PreScission protease before the in vitro ubiquitination assay
The mono- and poly-ubiquitination of A3H were performed by mixing 1 μM HA-A3H (and its variants)
0.4 μM E1 ubiquitin-activating enzyme (Enzo Life Sciences)
3.6 μM UBE2R1 (for poly-ubiquitination only)
1 mM DTT in 20 μl reaction volume and incubating for 2 h at room temperature
The reaction was stopped by adding 2x sample buffer
The ubiquitinated products were then subjected to western blot with anti-HA mAb from mouse (Sigma-Aldrich
5 µl of 0.02 mg/ml A3H-VCBCC complex sample was applied onto glow-discharged ultrathin formvar/carbon supported copper 400-mesh grids (EMS)
blotted and stained with 2.0% uranyl acetate
Negative-stained grids were imaged on a Tecnai F20 transmission electron microscope (FEI) operated at 200 kV
The freshly reconstituted protein complex was lightly crosslinked with 1 mM bis-sulfosuccinimidyl suberate (BS3
Thermo Fisher Scientific) on ice for 30 min
The reaction was quenched by 50 mM Tris (pH 8.0) for an additional 10 min
4 μl aliquots of 0.15 mg/ml purified A3H-VCBCC complex or A3H-VCBCCR complex were applied to graphene oxide-coated Quantifoil R1.2/1.3 gold 400-mesh grids (Electron Microscopy Sciences)
Grids were then blotted and vitrified in liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific)
Vitrified EM grids were screened in Talos F200C (Thermo Fisher Scientific) or Tecnai F20 (FEI) transmission electron microscopes to optimize the freezing conditions
Cryo-EM data of the A3H-VCBCC complex were collected in Titan Krios (Thermo Fisher Scientific) equipped with K3 direct electron detector and post-BioQuantum GIF energy filter (Gatan) operated at 300 kV in electron counting mode
Movies were collected at a nominal magnification of 165,000× and a pixel size of 0.51 Å
A total dose of 50 e−/Å2 per movie was used with a total exposure time of approximately 3.5 s
A total of 14,725 movies were recorded by automated data acquisition with EPU (Thermo Fisher Scientific)
Cryo-EM data of the A3H-VCBCCR complex were collected in Glacios (Thermo Fisher Scientific) equipped with Falcon-4 direct electron detector operated at 200 kV in electron counting mode
Movies were collected at a nominal magnification of 150,000× and a pixel size of 0.92 Å in EER format
A total dose of 40 e-/Å2 per movie was used with a dose rate of 5-6 e−/Å2/s
A total of 12,546 movies were recorded by automated data acquisition with EPU
an extra round of ab initio reconstruction and heterogenous refinement was performed to yield three classes
Two good classes from the heterogenous refinement were selected
and the particles were re-extracted with full resolution
Non-uniform refinement was then performed to yield the final 3.2 Å resolution map
Further information on research design is available in the <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41467-023-40955-x#MOESM7">Nature Portfolio Reporting Summary</a> linked to this article
Structure-guided analysis of the human APOBEC3-HIV restrictome
APOBEC3F properties and hypermutation preferences indicate activity against HIV-1 in vivo
The activity spectrum of Vif from multiple HIV-1 subtypes against APOBEC3G
Human cytidine deaminase APOBEC3H restricts HIV-1 replication
HIV-1 Vif adaptation to human APOBEC3H haplotypes
Family-wide comparative analysis of cytidine and methylcytidine deamination by eleven human APOBEC proteins
APOBEC3 multimerization correlates with HIV-1 packaging and restriction activity in living cells
subcellular localization and mC selectivity of a genomic mutator and anti-HIV factor APOBEC3H
The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by an RNA-Mediated Dimerization Mechanism
Structural basis of chimpanzee APOBEC3H dimerization stabilized by double-stranded RNA
APOBEC3H structure reveals an unusual mechanism of interaction with duplex RNA
The resistance of human APOBEC3H to HIV-1 NL4-3 molecular clone is determined by a single amino acid in Vif
The range of human APOBEC3H sensitivity to lentiviral Vif proteins
Natural polymorphisms in human APOBEC3H and HIV-1 Vif combine in primary T lymphocytes to affect viral G-to-A mutation levels and infectivity
Mapping region of human restriction factor APOBEC3H critical for interaction with HIV-1 Vif
Stably expressed APOBEC3H forms a barrier for cross-species transmission of simian immunodeficiency virus of chimpanzee to humans
Structural basis of intersubunit recognition in elongin BC-cullin 5-SOCS box ubiquitin-protein ligase complexes
The molecular basis of JAK/STAT inhibition by SOCS1
Polymorphisms and splice variants influence the antiretroviral activity of human APOBEC3H
A single amino acid difference in human APOBEC3H variants determines HIV-1 Vif sensitivity
Determinants of efficient degradation of APOBEC3 restriction factors by HIV-1 Vif
Two distinct types of E3 ligases work in unison to regulate substrate ubiquitylation
Structural insights into RNA bridging between HIV-1 Vif and antiviral factor APOBEC3G
Guidelines for naming nonprimate APOBEC3 genes and proteins
Genetic and mechanistic basis for APOBEC3H alternative splicing
Antiretroelement activity of APOBEC3H was lost twice in recent human evolution
Biochemical characterization of APOBEC3H variants: implications for their HIV-1 restriction activity and mC modification
The structural interface between HIV-1 Vif and human APOBEC3H
Lentiviral Vif degrades the APOBEC3Z3/APOBEC3H protein of its mammalian host and is capable of cross-species activity
Recombinant protein complex expression in E
Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs
Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction
High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy
UCSF Chimera-a visualization system for exploratory research and analysis
Towards automated crystallographic structure refinement with phenix.refine
<a data-track="click" data-track-action="download citation references" data-track-label="link" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/s41467-023-40955-x?format=refman&amp;flavour=references">Download references</a>
Electron microscopy data were collected at the Core Center of Excellence in Nano Imaging (CNI) at USC and California NanoSystems Institute (CNSI) at UCLA
which is supported in part by grants from the National Science Foundation (DBI-1338135 and DMR-1548924)
Cryo-EM data was computed at the Center for Advanced Research Computing (CARC) at USC
and John Curulli for assisting with the operation and maintenance of transmission electron microscopes at CNI
Tomek Osinski for assisting with computing work at CARC
and Cornelius Gati for advice on cryo-EM sample preparation and data processing
This work was funded by NIH grant R01AI150524 to X.S.C
was a former fellowship awardee from Nakajima Foundation
conceived the project and designed the experiments
purified the proteins and reconstituted the protein complex
performed the functional biochemical analyses
Nature Communications thanks Yasumasa Iwatani and the other anonymous reviewer(s) for their contribution to the peer review of this work
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DOI: https://doi.org/10.1038/s41467-023-40955-x
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Great effort has been devoted to discovering the basis of A3G-Vif interaction
the key event of HIV’s counteraction mechanism to evade antiviral innate immune response
Here we show reconstitution of the A3G-Vif complex and subsequent A3G ubiquitination in vitro and report the cryo-EM structure of the A3G-Vif complex at 2.8 Å resolution using solubility-enhanced variants of A3G and Vif
We present an atomic model of the A3G-Vif interface
which assembles via known amino acid determinants
This assembly is not achieved by protein-protein interaction alone
The cryo-EM structure and in vitro ubiquitination assays identify an adenine/guanine base preference for the interaction and a unique Vif-ribose contact
This establishes the biological significance of an RNA ligand
Further assessment of interactions between A3G
and RNA ligands show that the A3G-Vif assembly and subsequent ubiquitination can be controlled by amino acid mutations at the interface or by polynucleotide modification
suggesting that a specific chemical moiety would be a promising pharmacophore to inhibit the A3G-Vif interaction
Source data are provided as a Source data file
f Cryo-EM reconstruction of the sA3G-VC-RNA20 complex at 2.8 Å resolution
The complex is a C2-symmetric dimer (right)
The asymmetric unit (left) is colored in the same manner as in (b)
g Ribbon presentation of the atomic model of sA3G-VC-RNA20 complex and dissection of its components
The orientation and coloring scheme follow the right panel in (f)
Our cryo-EM structure shows interactions between A3G-Vif
and in vitro ubiquitination assays identify key adenine and guanine bases for the Vif-induced ubiquitination of A3G
Selected side chains and nucleotides are presented as stick models and labeled
Predicted hydrogen bonds between the ribose 2’-hydroxyl group and side chains H42/H43 of Vifred are represented by dashed lines (d)
f Stereo views of the atomic model showing nucleotide rA7 (e) and rA18 (f) accommodations in protein surface pockets of sA3G and Vifblue
Polypeptides and nucleotides are shown as stick models or in ribbon representation
Predicted hydrogen bonds between protein and nucleotides are depicted by cyan-colored dashed lines
It is noteworthy that Vifblue H43 and W70 contact sA3G Y19
whereas the same Vif residues face sA3G W127 and D128 in the sA3G-Vifred interface
These intermolecular interactions would compensate for an entropic penalty upon the complex assembly to confer base-specificity of the RNA ligand
The molecular arrangement of this pocket likely excludes the O6 atom of guanine
Adenine satisfies the geometry and interactions with chemical moieties of surrounding amino acids
nucleotide rC17 is exposed to the protein exterior
The atomic model shows that phosphate groups of rC17 and rA18 are within hydrogen bonding distance of side chains of T20 and R17
the impact of T32 phosphorylation on degradation is also likely caused by exclusion of the ligand RNA
Although these amino acids are located away from the sA3G-Vif interface
they mediate complex formation indirectly through interactions with RNA
Our cryo-EM map and biochemical data demonstrate that RNA mediates both sA3G-Vifred and sA3G-Vifblue assemblies
The RNA20 ligand increases the interface areas by up to ~1500 A2 and ~950 A2 for sA3G-Vifred-RNA20 and sA3G-Vifblue-RNA20
thus maximizing domain interactions and stabilizing the complex
It explains how RNA20 captures the sA3G-VC complex and enables structure determination by cryo-EM
participate in base-specific interactions with their protein binding partners
sA3G-VC assembly can be promoted by various RNA sequences
We further assessed base specificity and impact on sA3G ubiquitination
The RNA20 model is drawn in cartoon representation
binding sites on sA3G with Vifred and RNA20 are colored red and yellow
whereas interaction sites on Vifred with sA3G and RNA20 are colored green and yellow
sA3G interacts with Vifblue and RNA20 via regions colored blue and yellow
while Vifblue binds to sA3G and RNA20 through regions colored green and yellow
f–i Electrostatic potential surface representations of sA3G without (f) and with (g) RNA20
Calculated surface potentials are colored with a gradient from red (negative) to blue (positive)
The electrostatic potential scale is indicated in (i)
</a>a In vitro ubiquitination assay using sA3G and its mutants
Substrates were specifically detected using a C-terminally-tagged c-myc sequence (lanes 1 and 2)
Ubiquitination of sA3G required ARIH2 and ubiquitin with a C-terminal G76 (lanes 3 and 4)
The reaction was enhanced in the presence of RNA20 (compare lanes 5 and 8)
D128K (lane 6) and K4Rs (K297R/K301R/K303R/K334R) (lane 7) appeared to reduce polyubiquitination beyond diubiquitin
Most of sA3G was ubiquitinated within 1 h of reaction (lane 9)
The band of unreacted sA3G is indicated by an arrow
sA3G was depleted within 30 min (lanes 1–4)
whereas the ubiquitination was attenuated by sA3G mutation D128K (lanes 5–8)
DNA20 showed almost no sA3G ubiquitination (lanes 9–12)
c Quantification of unreacted sA3G during the reaction presented in (b)
Assays were performed independently in triplicate (n = 3)
e Impact of dinucleotide rG6rA7 on sA3G ubiquitination
Reactants were analyzed by western blotting (d)
The amount of unreacted sA3G was quantified (e)
A significant (p &lt; 0.05 by two-sided t test
no adjustments) decrease in unreacted sA3G is indicated by an asterisk compared with that in presence of U20; p values are 0.0095
sA3G was depleted in the presence of U20-rGrA (d
whereas U20 showed a much slower reaction (d
U20-rUrA retained some ability to enhance sA3G ubiquitination (d
lanes 9–12) and U20-rGrU (e) significantly lost the ability to enhance sA3G ubiquitination
f Close-up view of dinucleotide rG6rA7 at the sA3G-Vifred interface
The solvent-accessible surface of sA3G is shown in green
and side chains of Vifred H42/H43 are drawn in stick representation and labeled
Predicted hydrogen bonds are depicted as dashed yellow lines
Source data are provided as a Source data file (a–e)
The molecular models show that U9-rGrA and U7-rGrA are too short to provide a large negatively charged surface for Vif interaction
our in vitro ubiquitination and in cell degradation assays showed that the dinucleotide rG6rA7 serves an essential biological function in conferring local base preference upon the RNA ligand to promote A3G-Vif interaction
The length of RNA ligand is likely important for the enhancement of the A3G-Vif interaction
it is plausible that A3G-Vif and A3G-Gag interactions may compete with each other and reduce encapsidation of A3G
</a>a, b Comparison of Vif-binding interfaces from sA3G (a) (this study) and A3F (b)<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 40" title="Hu, Y. et al. Structural basis of antagonism of human APOBEC3F by HIV-1 Vif. Nat. Struct. Mol. Biol. 26, 1176–1183 (2019)." href="/articles/s41467-023-39796-5#ref-CR40" id="ref-link-section-d455805266e2135">40</a>
The molecular orientation of A3 proteins is indicated using a cartoon representation (bottom right inset)
whereas the interface on A3F with a single Vif is colored red (b)
c Predicted model of the entire ubiquitin ligase complex
The model was built using alignments of atomic coordinates obtained from our cryo-EM structure and PDB IDs 4N9F
d Solvent-accessible surface representations of the predicted ubiquitin ligase complex linked through sA3G-Vifred-CUL5
Amino acids sA3G K297/K301/K303/K334 and ubiquitin G75 are drawn as space-filled models and labeled
e Unprocessed TEM micrograph of a negatively stained sample from a mixture of in vitro ubiquitination assay reactant
The image reflects many states of the ongoing reaction
Arrowheads indicate possible U-shaped ubiquitin ligase complex particles
f 2D class average images of putative U-shaped ubiquitin ligase complex particles
The 6 classes were composed of 1095 particles harvested from 179 micrographs
g Superimposition of the predicted ubiquitin ligase complex model (d) on a class average image from (f)
our high-resolution cryo-EM structure of sA3G-VC-RNA20 revealed that Vif binds sA3G through interactions with ssRNA
Our in vitro and in cellulo experiments showed that the sA3G-Vif-RNA trimolecular interface involving the dinucleotide rG6rA7 is most critical for ubiquitination of sA3G; therefore
this trimolecular interface is likely used to form a ubiquitin E3-ligase complex
The good fit of the predicted ubiquitin ligase complex model with 2D class average images holds promise for determining the structure of the fully assembled ubiquitin ligase complex
which may provide further understanding of structure-activity relationships of A3G ubiquitination
Preparation procedures were partially modified and are briefly described as follows
Expression plasmids for glutathione S-transferase (GST)-fused sNTD or sA3G and its mutants were constructed using pCold-GST vectors (a gift from Dr
coli strain BL21 (DE3) was transformed using the plasmid and cultivated in LB media at 37 °C
expression was induced at 20 °C by adding 0.2 mM isopropyl-β-thiogalactopyranoside (IPTG)
cells were harvested and resuspended in buffer-1 [50 mM sodium phosphate
and 0.5-mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)]
and the lysate was centrifuged at 20,000 × g for 30 min
The supernatant was applied to glutathione-immobilized resin (Genscript) equilibrated in buffer-1
The protein-bound resin was washed with buffer-2 (50 mM Tris-HCl
An aliquot of HRV 3 C protease solution (Fujifilm Wako Pure Chemical
Japan) was added to the resin resuspension
and the mixture was incubated at 4 °C overnight
The supernatant was applied to a Superdex 200 column (Cytiva) equilibrated with buffer-3 (25 mM Tris-HCl
Fractionated protein was concentrated and kept in a freezer at −80 °C until further use
Protein purity was verified on 12% or 15% polyacrylamide gels followed by Coomassie brilliant blue staining
or on 4–12% gradient Bis-Tris gels (Invitrogen) stained with SimplyBlue SafeStain (Invitrogen)
sA3G mutants and C-terminally c-myc-tagged constructs were prepared in the same manner
Vif and CBFβ constructs shown in Fig.&nbsp;<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41467-023-39796-5#Fig1">1b</a> were inserted into BamHI/HindIII and NdeI/XhoI sites
coli strain BL21 (DE3) was transformed using this plasmid and cultivated in LB media at 37 °C
cells were harvested and resuspended in buffer-2 supplemented with 20 mM imidazole and 10% dimethyl sulfoxide (DMSO)
The supernatant was applied to buffer-2-equilibrated Ni-NTA resin (Fujifilm Wako Pure Chemical)
bound proteins were eluted with buffer-2 supplemented with 200 mM imidazole and 500 mM dimethylethylammonium propane sulfonate
Eluent was loaded onto a Superdex 200 column equilibrated in buffer 2
and the mixture was concentrated and kept in a freezer at −80 °C until further use
ubiquitin-conjugating enzyme E2L3 (M1 to D154)
NEDD8-conjugating enzyme UBE2F (M1 to R185) were codon-optimized for bacterial expression and synthesized (Genscript)
Their mutants were constructed using a PCR-based technique
and UBE2F were individually inserted into NdeI/XhoI sites on pCold-GST vectors
Protein expression and purification followed the procedure for sA3G preparation described above
DNAs encoding CUL5 and Rbx2 constructs were inserted into the BamHI/XhoI site on a pGEX-6P-1 vector (Cytiva)
and into BamHI/HindIII sites on a pRSFDuet vector (Novagen)
Proteins CUL5 and Rbx2 were co-expressed and co-purified in a manner similar to sA3G preparation
DNAs encoding NAE1 and UBA3 were inserted into BamHI/HindIII and NdeI/XhoI sites
NAE1/UBA3 preparation followed the method for VC purification described above
Obtained protein solutions were concentrated and then frozen at −80 °C until further use
Residue numbering follows wild-type constructs
The fraction of sNTD-F126 in complex with VCBC was prepared for RNA extraction with TRIzol and precipitated with ethanol
The pellet was used for a serial enzymatic reaction and for the cloning: 3′-end of RNA was extended by poly(A) polymerase (New England BioLabs)
5′-adapter (5′-rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrC-3′) was ligated with T4 RNA ligase (New England BioLabs)
followed by reverse transcription with a poly-thymidine DNA oligo
and amplification of the cDNA by polymerase chain reaction (PCR) using Taq DNA polymerase (Takara Bio
Resulting DNAs were inserted into pMD20-T vectors (Takara Bio
and clones were subjected to DNA sequencing
Purified sA3G, VC and a synthesized RNA oligomer (see Table&nbsp;<a data-track="click" data-track-label="link" data-track-action="table anchor" href="/articles/s41467-023-39796-5#Tab1">1</a>; IDT technologies
Japan) were mixed at a ratio of 1:1.5:1.5 in buffer-3 supplemented with 200 mM NDSB-195
The mixture was applied to a Superdex 200 column equilibrated in buffer-3 and purified by size-exclusion chromatography
The largest fraction of the complex was used for negative staining EM and cryo-EM grid preparations immediately after collection
The relative amount of harvested complex was monitored by UV absorbance at 280 nm (A280)
and RNA binding was roughly estimated by the A260/A280 ratio
The prepared sA3G-VC-RNA complex typically had a ratio of 0.98–1.02
and VCBC were mixed at a final concentration of 5 μM each
with 10 μM NEDD8 and 1 mM of freshly prepared ATP in an assay buffer
The mixture was incubated at 20 °C for at least 1 h
NEDDylation of CUL5 was verified by PAGE analysis
A 10-μL aliquot of the resulting mixture was mixed with 1 μM sA3G or its mutant
and the total volume was adjusted to 50 μL with assay buffer
and an aliquot of the reactant was harvested for further analysis after 0
The concentration of freshly prepared sA3G-VC-RNA complex solution was adjusted so that the UV absorbance at 280 nm was in the range of 0.30–0.33
A holey EM grid (Quantifoil R1.2/1.3) was plasma-cleaned (Solarus II
Gatan Inc.) and treated with graphene oxide film flakes (Sigma Aldrich)
3 μL of sample solution was deposited onto the grid at 4 °C and 100% humidity
The sample grid was blotted with filter paper for 3 s and vitrified by plunging it into ethane/propane at liquid nitrogen temperature on a Vitrobot Mark IV (Thermo Fisher Scientific)
Vitrified sample grids were stored in liquid nitrogen until further use
All cryo-EM data were collected semi-automatically using EPU software (Thermo Fisher Scientific)
Electrostatic potentials were calculated with the program
statistical tests were conducted to evaluate the statistical significance between two datasets: F-test for evaluation of standard deviation equivalency and t-test for assessment of significant difference between two data sets
The significant difference was defined as less than 0.05 of the p value
Source data and uncropped unprocessed scans are provided as a Source data file
Further information on research design is available in the&nbsp;<a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41467-023-39796-5#MOESM3">Nature Portfolio Reporting Summary</a> linked to this article
Structural perspectives on HIV-1 Vif and APOBEC3 restriction factor interactions
Suppression of APOBEC3-mediated restriction of HIV-1 by Vif
APOBEC3G DNA deaminase acts processively 3’ -&gt; 5’ on single-stranded DNA
The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA
Cone-shaped HIV-1 capsids are transported through intact nuclear pores
Vif Hijacks CBF-β to degrade APOBEC3G and promote HIV-1 infection
Structural basis for hijacking CBF-β and CUL5 E3 ligase complex by HIV-1Vif
Defining APOBEC3 expression patterns in human tissues and hematopoietic cell subsets
Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G
Definition of the interacting interfaces of Apobec3G and HIV-1 Vif using MAPPIT mutagenesis analysis
Amino-terminal region of the human immunodeficiency virus type 1 nucleocapsid is required for human APOBEC3G packaging
Specific packaging of APOBEC3G into HIV-1 virions is mediated by the nucleocapsid domain of the gag polyprotein precursor
Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is incorporated into HIV-1 virions through interactions with viral and nonviral RNAs
APOBEC3G incorporation into human immunodeficiency virus type 1 particles
Complementary function of the two catalytic domains of APOBEC3G
catalytically active human APOBEC3G: correlation with antiviral effect
Crystal structure of the catalytic domain of HIV-1 restriction factor APOBEC3G in complex with ssDNA
Computational model and dynamics of monomeric full-length APOBEC3G
High-speed atomic force microscopy directly visualizes conformational dynamics of the HIV Vif protein in complex with three host proteins
The HIV-1 Vif PPLP motif is necessary for human APOBEC3G binding and degradation
Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity
Insights into DNA substrate selection by APOBEC3G from structural
Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation
Promiscuous RNA binding ensures effective encapsidation of APOBEC3 proteins by HIV-1
NIH Image to ImageJ: 25 years of image analysis
CTFFIND4: fast and accurate defocus estimation from electron micrographs
Macromolecular structure determination using X-rays
neutrons and electrons: recent developments in Phenix
MolProbity: more and better reference data for improved all-atom structure validation
Scalable molecular dynamics on CPU and GPU architectures with NAMD
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This research was supported by the Platform Project for Supporting Drug Discovery and Life Science Research (BINDS) from AMED under grant number JP18am0101076 and by direct funding from OIST (to M.W.); the Toyobo Biotechnology Foundation (to T.K.) and T.K.‘s personal funds
was supported in part by a grant from the U.S
National Institutes of Health R01GM118474/R01AI150478 and federal funds from the National Cancer Institute
Chojiro Kojima at Osaka University for providing pCold-GST vectors
Keiko Kono at OIST for advice on human cell experiments and western blotting analysis
Melissa Matthews for critical reading of the manuscript
Aird for technical editing of the manuscript
We thank the OIST Imaging and Analysis Section (IMG) for use of the EM facility
and the OIST Scientific Data Analysis Section (SCDA) for use of the Deigo and Saion high performance computing clusters
Adrian Koh for collecting cryo-EM test data of sA3G-VC-RNA-IV-20 on the Titan Krios G4 Selectris-X at Thermo Fisher Scientific Eindhoven
Present address: Department of Efficacy Evaluation
Okinawa Institute of Science and Technology Graduate University
Frederick National Laboratory for Cancer Research
designed sA3G and all wild-type A3G/sA3G mutants
designed all DNA/RNA/hybrid oligomer sequences
cloned all protein constructs used in this study
prepared grapheneoxide-coated cryo-EM grids
processed cryo-EM data with support of S.S.
conducted atomic model building and refinement
analyzed molecular structures and recapitulated the entire project
All authors read and contributed to writing the manuscript
supervised and provided guidance on all cryo-EM experiments
has filed patents on preparation of the solubility-enhanced human A3G construct
its application for A3G-Vif complex analysis [PCT-JP2019-019938 (2019) and JP patent application number 2021-519919 (2021)]; on DNA/RNA/hybrid-based antagonism against A3G-Vif interaction [PCT-JP2021-42773 (2021)]
Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work
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DOI: https://doi.org/10.1038/s41467-023-39796-5
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HIV-1 virion infectivity factor (Vif) promotes degradation of the antiviral APOBEC3 (A3) proteins through the host ubiquitin-proteasome pathway to enable viral immune evasion
Disrupting Vif-A3 interactions to reinstate the A3-catalyzed suppression of human immunodeficiency virus type 1 (HIV-1) replication is a potential approach for antiviral therapeutics
the molecular mechanisms by which Vif recognizes A3 proteins remain elusive
Here we report a cryo-EM structure of the Vif-targeted C-terminal domain of human A3F in complex with HIV-1 Vif and the cellular cofactor core-binding factor beta (CBFβ) at 3.9-Å resolution
The structure shows that Vif and CBFβ form a platform to recruit A3F
revealing a direct A3F-recruiting role of CBFβ beyond Vif stabilization
and captures multiple independent A3F-Vif interfaces
Together with our biochemical and cellular studies
our structural findings establish the molecular determinants that are critical for Vif-mediated neutralization of A3F and provide a comprehensive framework of how HIV-1 Vif hijacks the host protein degradation machinery to counteract viral restriction by A3F
Other data are available from corresponding authors upon reasonable request
T-cell differentiation factor CBF-β regulates HIV-1 Vif-mediated evasion of host restriction
Evolution of the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases
Identification of specific determinants of human APOBEC3F
and APOBEC3DE and African green monkey APOBEC3F that interact with HIV-1 Vif
The retroviral hypermutation specificity of APOBEC3F and APOBEC3G is governed by the C-terminal DNA cytosine deaminase domain
Core binding factor beta plays a critical role by facilitating the assembly of the Vif-cullin 5 E3 ubiquitin ligase
CBFβ stabilizes HIV Vif to counteract APOBEC3 at the expense of RUNX1 target gene expression
Structural basis for hijacking CBF-β and CUL5 E3 ligase complex by HIV-1 Vif
Crystal structure of the DNA cytosine deaminase APOBEC3F: the catalytically active and HIV-1 Vif-binding domain
Structural insights into HIV-1 Vif-APOBEC3F interaction
Identification of 81LGxGxxIxW89 and 171EDRW174 domains from human immunodeficiency virus type 1 Vif that regulate APOBEC3G and APOBEC3F neutralizing activity
The APOBEC3C crystal structure and the interface for HIV-1 Vif binding
A single amino acid in human APOBEC3F alters susceptibility to HIV-1 Vif
Transcriptional regulation of APOBEC3 antiviral immunity through the CBF-β/RUNX axis
The assembly of Vif ubiquitin E3 ligase for APOBEC3 degradation
The binding interface between human APOBEC3F and HIV-1 Vif elucidated by genetic and computational approaches
Multifaceted counter-APOBEC3G mechanisms employed by HIV-1 Vif
HIV-1 viral infectivity factor (Vif) alters processive single-stranded DNA scanning of the retroviral restriction factor APOBEC3G
Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation
Phosphorylation of a novel SOCS-box regulates assembly of the HIV-1 Vif-Cul5 complex that promotes APOBEC3G degradation
Evolutionary paradigms from ancient and ongoing conflicts between the lentiviral Vif protein and mammalian APOBEC3 enzymes
Evolution of HIV-1 isolates that use a novel Vif-independent mechanism to resist restriction by human APOBEC3G
Long-term restriction by APOBEC3F selects human immunodeficiency virus type 1 variants with restored Vif function
HIV-1 and HIV-2 Vif interact with human APOBEC3 proteins using completely different determinants
Codon optimization of the HIV-1 vpu and vif genes stabilizes their mRNA and allows for highly efficient Rev-independent expression
Generation of high-titer pseudotyped retroviral vectors with very broad host range
Cytokine signals are sufficient for HIV-1 infection of resting human T lymphocytes
Identification of a tripartite interaction between the N-terminus of HIV-1 Vif and CBFβ that is critical for Vif function
Gctf: real-time CTF determination and correction
Prevention of overfitting in cryo-EM structure determination
UCSF Chimera—a visualization system for exploratory research and analysis
REFMAC5 for the refinement of macromolecular crystal structures
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Devarkar and other Xiong lab members for discussions
This work was supported by National Institutes of Health grant AI116313 (Y.X.)
and by an Intramural AIDS Targeted Antiviral Program grant and the Innovation Fund
Present address: Department of Biochemistry and Biophysics
The authors declare no competing financial interests
Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team
The Vif–CBFβ–A3FCTDm fusion complex with or without the Vif α-domain and corresponding interacting CBFβ C terminus stays as a tetramer in low-salt solution
The unfused Vif–CBFβ–A3FCTDm without these regions switches from monomer to tetramer at high protein concentration (146 μΜ loading concentration)
No obvious shift for the elution peak was observed upon incubation of CBFβ and A3FCTDm compared to the CBFβ alone or A3FCTDm alone
The SDS-PAGE analysis of the peak fractions of CBFβ alone
A3FCTDm alone and CBFβ/A3FCTDm mixture is indicated
Co-immunoprecipitation (Co-IP) analysis of the interaction between A3F and CBFβ in the presence or absence of Vif in cells
Flag-A3F and CBFβ-myc were cotransfected with or without Vif-HA and co-immunoprecipitated using an anti-Flag antibody
no binary A3F and CBFβ binding was observed
A representative blot from two independent experiments was shown
The 5-Å cryo-EM reconstruction of Vif–CBFβ–A3FCTDm with (right) or without (left) the docked-in model (ribbon)
The density corresponding to the flexible Vif α-domain and the corresponding interacting CBFβ C terminus (circled) is not visible
the 3.9-Å cryo-EM reconstruction of the truncated Vif–CBFβ–A3FCTDm
overlay of the cryo-EM models of Vif–CBFβ–A3FCTDm ternary complexes with (magenta) and without (yellow) the Vif α-domain and the corresponding interacting CBFβ C terminus shows that the removal of these regions does not affect the architecture of the ternary complex
Central slices of the top 3D classes of the Vif–CBFβ–A3FCTDm complex with (upper) or without (lower) the Vif α-domain and the corresponding interacting CBFβ C terminus indicate that removing these flexible regions reduces the tetramer flexibility
The location of the Vif α-domain and the corresponding interacting CBFβ C terminus is marked by yellow arrows in the first class average
The color spectrum and the coil thickness represent the deviation of the aligned Cα atoms in the structures, which varies from 0 Å (blue) to ~10 Å (orange). The Vif C-terminal residues 173–176 missing in the Vif–E3 ligase structure are colored in red. The Vif–CBFβ structure without A3FCTDm binding used for superposition is extracted from the Vif–E3 ligase structure (PDB <a href="https://www.rcsb.org/structure/4N9F">4N9F</a>)
The effect of D347R mutation on A3F sensitivity to Vif-mediated degradation indicated by western blot (left)
quantified A3F levels relative to no Vif (mean ± s.d.; n = 4 biologically independent experiments; middle)
and relative infectivity (mean ± s.d.; n = 4 biologically independent experiments; right)
A3FD347R retained wild-type A3F-like sensitivity to Vif-mediated degradation
which resulted in rescue of viral infectivity
The effect of D260A/D261A or D260R/D261R double mutants on A3F sensitivity to Vif-mediated degradation indicated by western blot (left)
quantified A3F levels relative to no Vif (mean ± s.d.; n = 3 biologically independent experiments; middle)
and relative infectivity (mean ± s.d.; n = 3 biologically independent experiments; right)
Both alanine and arginine mutants did not confer resistance
indicating that the side chains of the residues are not involved in the Vif interaction
Western blot (a) and quantified A3 levels relative to ‘no Vif’ (b) show that the Vif K50E mutant could not induce A3F degradation in the presence of either CBFβ wild-type or CBFβ E54K but could induce A3G degradation (mean ± s.d.; n = 3 biologically independent experiments)
the charge-swapped Vif R15E/A3F E289K double mutation did not restore the Vif-mediated A3F degradation or viral infectivity in cells
The blot was cut as indicated (gray arrow)
where one half was used to detect Flag-A3F and HSP90
and the other half was used to detect Vif-HA
left) blocks the catalytic site of A3FCTDm (red and marked by an arrow)
One Vif–CBFβ–A3FCTDm ternary complex with the major interface is circled with an oval
FSC curves of the half-maps from gold standard refinements of the Vif–CBFβ–A3FCTDm complex with (cyan) or without (blue) the Vif α-domain and the corresponding interacting CBFβ C terminus
The FSC curve of the map and final model of the truncated Vif–CBFβ–A3FCTDm complex is in green
Resolution of the maps are determined by the cut-off values at FSC = 0.143
The Euler angle distribution of the classified particles of the truncated Vif–CBFβ–A3FCTDm complex used for the final 3D reconstruction
Color coded local resolution estimation of the D2 symmetrized map of the truncated Vif–CBFβ–A3FCTDm complex
a−c, Detailed illustrations of the secondary structure elements of Vif176 (a), CBFβ151 (b) and A3FCTD (c). The secondary structures are annotated on primary amino acid sequences (left) and tertiary structures (right). The tertiary structures for illustration are: Vif, extracted from PDB <a href="https://www.rcsb.org/structure/4N9F">4N9F</a>; CBFβ151, extracted from our cryo-EM structure; A3FCTD, PDB <a href="https://www.rcsb.org/structure/3WUS">3WUS</a>
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DOI: https://doi.org/10.1038/s41594-019-0343-6
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HIV-1 infection-induced cGAS–STING–TBK1–IRF3 signaling activates innate immunity to produce type I interferon (IFN)
The HIV-1 nonstructural protein viral infectivity factor (Vif) is essential in HIV-1 replication
as it degrades the host restriction factor APOBEC3G
whether and how it regulates the host immune response remains to be determined
we found that Vif inhibited the production of type I IFN to promote immune evasion
HIV-1 infection induced the activation of the host tyrosine kinase FRK
which subsequently phosphorylated the immunoreceptor tyrosine-based inhibitory motif (ITIM) of Vif and enhanced the interaction between Vif and the cellular tyrosine phosphatase SHP-1 to inhibit type I IFN
the association of Vif with SHP-1 facilitated SHP-1 recruitment to STING and inhibited the K63-linked ubiquitination of STING at Lys337 by dephosphorylating STING at Tyr162
the FRK inhibitor D-65495 counteracted the phosphorylation of Vif to block the immune evasion of HIV-1 and antagonize infection
These findings reveal a previously unknown mechanism through which HIV-1 evades antiviral immunity via the ITIM-containing protein to inhibit the posttranslational modification of STING
These results provide a molecular basis for the development of new therapeutic strategies to treat HIV-1 infection
Carbohydrate-specific signaling through the DC-SIGN signalosome tailors immunity to Mycobacterium tuberculosis
HIV-1 exploits innate signaling by TLR8 and DC-SIGN for productive infection of dendritic cells
Type I interferon responses by HIV-1 infection: association with disease progression and control
Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8
HIV-1 single-stranded RNA induces CXCL13 secretion in human monocytes via TLR7 activation and plasmacytoid dendritic cell-derived type I IFN
Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses
IFI16 senses DNA forms of the lentiviral replication cycle and controls HIV-1 replication
The capsids of HIV-1 and HIV-2 determine immune detection of the viral cDNA by the innate sensor cGAS in dendritic cells
IFI16 DNA sensor is required for death of lymphoid CD4 T cells abortively infected with HIV
Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA
Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway
Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA
NONO detects the nuclear HIV capsid to promote cGAS-mediated innate immune activation
IFI16 targets the transcription factor Sp1 to suppress HIV-1 transcription and latency reactivation
Structural basis of STING binding with and phosphorylation by TBK1
Interferon responses in HIV infection: from protection to disease
Type I interferon and HIV: subtle balance between antiviral activity
The macrophage in HIV-1 infection: from activation to deactivation
The cytokine network of acute HIV infection: a promising target for vaccines and therapy to reduce viral set-point
Cyclophilin A modulates the sensitivity of HIV-1 to host restriction factors
CPSF6 defines a conserved capsid interface that modulates HIV-1 replication
HIV-1 evades innate immune recognition through specific cofactor recruitment
Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu
HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation
Restricting HIV the SAMHD1 way: through nucleotide starvation
Restriction by SAMHD1 limits cGAS/STING-dependent innate and adaptive immune responses to HIV-1
HIV-1 blocks the signaling adaptor MAVS to evade antiviral host defense after sensing of abortive HIV-1 RNA by the host helicase DDX3
NLRX1 sequesters STING to negatively regulate the interferon response
thereby facilitating the replication of HIV-1 and DNA viruses
HIV-1 and interferons: who’s interfering with whom
The cytosolic exonuclease TREX1 inhibits the innate immune response to human immunodeficiency virus type 1
Immunoreceptor tyrosine-based inhibition motifs: a quest in the past and future
Inhibition of TLR signaling by a bacterial protein containing immunoreceptor tyrosine-based inhibitory motifs
The E3 ligases Itch and WWP2 cooperate to limit TH2 differentiation by enhancing signaling through the TCR
The ubiquitin ligase TRIM56 regulates innate immune responses to intracellular double-stranded DNA
TRIM32 protein modulates type I interferon induction and cellular antiviral response by targeting MITA/STING protein for K63-linked ubiquitination
TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity
The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination
The tyrosine kinase FRK/RAK participates in cytokine-induced islet cell cytotoxicity
Innate immune sensing of HIV-1 by dendritic cells
Shp1 regulates T cell homeostasis by limiting IL-4 signals
Activation of phosphotyrosine phosphatase activity attenuates mitogen-activated protein kinase signaling and inhibits c-FOS and nitric oxide synthase expression in macrophages infected with Leishmania donovani
SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-kappaB and interferon pathways
Structural insight into the human immunodeficiency virus Vif SOCS box and its role in human E3 ubiquitin ligase assembly
The Phyre2 web portal for protein modeling
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This work was supported by grants from the Program of Shanghai Academic Research Leader (21XD1402900)
the Natural Science Foundation of Shanghai (21ZR1481400)
the National Natural Science Foundation of China (31972900)
the National Youth Talent Support Program (Ten Thousand Talent Program)
the National Key Research and Development Program of China (2018YFC1705505)
and the National Megaproject on Key Infectious Diseases (2017ZX10202102)
Sun (Shanghai Institute of Biochemistry and Cell Biology
Germany) for pBR322-HIV-1-M-NL4-3-IRES-eGFP env STOP plasmid (pseudotyping is required for infection)
China) for cDNAs encoding SRC-family kinases
These authors contributed equally: Yu Wang
Shanghai Institute of Infectious Disease and Biosecurity &amp; Shanghai Public Health Clinical Center
National Engineering Research Centre of Immunological Products
Department of Microbiology and Biochemical Pharmacy
National Engineering Laboratory for AIDS Vaccine
Shanghai Key Laboratory of Organ Transplantation
Zhongshan Hospital &amp; Institutes of Biomedical Sciences
and DY conceived the project and designed the experiments
and ZZ performed most of the experiments and analyzed the data
and TX assisted with the experiments and provided technical help
and XZ provided comments and assisted with manuscript preparation
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DOI: https://doi.org/10.1038/s41423-021-00802-9
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Accessory proteins are a key feature that distinguishes primate immunodeficiency viruses such as human immunodeficiency virus type I (HIV-1) from other retroviruses
A prime example is the virion infectivity factor
which hijacks a cellular co-transcription factor (CBF-&#x03B2;) to recruit a ubiquitin ligase complex (CRL5) to bind and degrade antiviral APOBEC3 enzymes including APOBEC3D (A3D)
Although APOBEC3 antagonism is essential for viral pathogenesis
and a more than sufficient functional justification for Vif&#x2019;s evolution
most viral proteins have evolved multiple functions
Vif has long been known to trigger cell cycle arrest and recent studies have shed light on the underlying molecular mechanism
Vif accomplishes this function using the same CBF-&#x03B2;/CRL5 ubiquitin ligase complex to degrade a family of PPP2R5 phospho-regulatory proteins
These advances have helped usher in a new era of accessory protein research and fresh opportunities for drug development
HIV-1 genome organization and counteraction of host-cell restriction factors by viral accessory proteins
(A) Depiction of the genomic organization of HIV-1 with yellow indicating structural proteins and viral enzymes
green indicating the envelope glycoprotein
and red highlighting the viral accessory proteins
(B) Simplified schematic of the HIV-1 life-cycle with activities of the viral accessory proteins highlighted in red
Nef and Vpu downmodulate the anti-viral factors TETHERIN and SERINC3/5 from the cell surface
Vif targets cytoplasmic APOBEC3s for proteasomal degradation prior to viron encapsidation
Vpr antagonizes many different cellular processes including DNA-damage response
APOBEC3 restriction and counter action by Vif
(A) Schematic of the APOBEC3 locus with anti-HIV-1 activity indicated above or below the respective APOBEC3 enzyme
(B) Depiction of APOBEC3 C-to-T mutations during conversion of the viral genomic RNA (gRNA) to cDNA
Mutations are depicted at either &#x201C;CC&#x201D; or &#x201C;TC&#x201D; dinucleotide contexts which is reflective of A3G or A3D/F/H target sites
Fixation of the mutations is depicted by the final double-stranded DNA product (right)
(C) Structure of the E3-ubiquitin ligase complex nucleated by Vif (PDB: 4N9F)
Full length CUL5 has been modeled in using the partial CUL5 sequence from PDB: 4N9F
RBX2 has been modeled in using RBX1 from PDB: 1LDJ as a template
The inset depicts the Vif/CBF-&#x03B2; hetero-dimer as ribbon diagrams from the crystal structure
(D) Amino acid sequences and surface models of Vif with substrate binding residues highlighted in the indicated color
The shared surface indicated residues that are required for degradation of two or more Vif substrates
these biochemical constraints suggest that a single Vif/E3-complex can only degrade one substrate at a time
making Vif&#x2019;s ability to target so many distinct substrates even more impressive
While these observations collectively pointed toward Vif degrading a cellular factor to induce arrest
the identity of this factor remained elusive for nearly 10 years following the discovery of this activity
The PPP2R5 surface recognized by Vif is extremely electronegative and supports a model in which Vif maintains this interaction through a favorable network of electrostatics
all of the residues required for Vif recognition are conserved among all five family members
which clarifies how Vif can recognize and degrade five new cellular substrates and explains genetic evidence that loss of at least two family members is required for inducing G2/M arrest
(A) Amino acid residues on PPP2R5 and APOBEC3 that have been shown to be required for Vif-mediated degradation (top) with substrate surfaces (middle) and electrostatic potential maps (bottom)
(B) Depiction of amino acid residues on the surface of PPP2R5 that are recognized by Vif or cellular substrates
The LxxIxE substrate peptide is depicted bound to the PPP2R5 surface to highlight that Vif and cellular substrates compete for an overlapping surface
these observations support a competitive binding model in which Vif directly interacts with the surface of PPP2R5 proteins and occludes the binding of cellular substrates
The discovery of PPP2R5 substrates was a major step forward in understanding Vif-induced cell cycle arrest
the regulatory checkpoints altered downstream of PPP2R5 degradation remain unknown
PP2A/PPP2R5 complexes have been shown to regulate entrance and exit of the G2-to-M phase transition at multiple different checkpoints
we discuss these checkpoints in the context of previous observations regarding Vif-induced G2/M arrest and postulate a model in which antagonism of discrete PP2A/PPP2R5 complexes would lead to simultaneous inhibition of multiple checkpoints
Diagram of potential Vif-induced G2/M arrest mechanisms
Depiction of normal and aberrant regulation of the G2-to-M phase transition in the absence (top) and presence (bottom) of Vif
Key cell cycle checkpoints regulated by PP2A/PPP2R5 complexes are color-coded in the absence or presence of Vif
In addition to manipulation of the PP2A/PPP2R5 axis, it has been suggested that Vif can induce G2/M arrest by blocking MDM2-mediated ubiquitination and nuclear export of TP53 (<a href="#B51">Izumi et al., 2010</a>)
This study observed that Vif binding to TP53 could block MDM2 recognition and subsequent turnover of TP53
which is required for proper cell cycle progression
it is likely that the Vif/TP53 interaction occurs in the nuclear compartment
thus directly shielding TP53 from MDM2 recognition and subsequent nuclear export
it is plausible that there are two pools of Vif that act in concert to stall cell cycle progression
Nuclear localized Vif protects TP53 from MDM2 and antagonizes nuclear PPP2R5C/D substrates
whereas cytoplasmic Vif antagonizes the APOBEC3s and cytoplasmic PPP2R5A/B/E
Additional work will be required to determine if these mechanisms are separate or connected through shared components
Vif clearly has two distinct sets of cellular substrates
APOBEC3 enzymes and PPP2R5 phospho-regulators (10 proteins total)
While APOBEC3 counteraction has been shown to be essential for viral infectivity and pathogenesis in vivo
the importance of PPP2R5 degradation remains to be established
at least three key observations have been made that imply that alteration of the cell cycle may be beneficial for HIV-1 pathogenesis
it is possible that Vif antagonism of PP2A holoenzymes leads to increased protein translation during G2/M when host cell translation is normally stalled
it is possible that Vif and Vpr act in concert to induce G2/M arrest and boost transcription and translation of HIV-1 genes
these observations support a model in which PPP2R5 antagonism
and global changes in the cellular phospho-proteome
are likely to be advantageous for the pathogenesis of HIV-1 as well as other prominent viruses
continuing to unravel the complex molecular mechanisms HIV-1 has evolved to subvert cellular processes and enhance pathogenicity may provide major clues for the development of innovative therapeutics that lead to virus eradication
Both authors contributed to the article and approved the submitted version
This work was supported by NIAID R37-AI064046 (to RH)
DS received salary support from an NIAID K99/R00 transition award (K99-AI147811)
RH is the Margaret Harvey Schering Land Grant Chair for Cancer Research
a Distinguished University McKnight Professor
and an Investigator of the Howard Hughes Medical Institute
and consultant of ApoGen Biotechnologies Inc
The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest
HIV-1 Vpr: mechanisms of G2 arrest and apoptosis
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Citation: Salamango DJ and Harris RS (2021) Dual Functionality of HIV-1 Vif in APOBEC3 Counteraction and Cell Cycle Arrest
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*Correspondence: Reuben S. Harris, <a id="encmail">cnNoQHVtbi5lZHU=</a>; Daniel J. Salamango, <a id="encmail">ZGFuaWVsLnNhbGFtYW5nb0BzdG9ueWJyb29rLmVkdQ==</a>
&#x2020;Present address: Daniel J. Salamango, Department of Microbiology and Immunology, Stony Brook University, Stony Brook, NY, United States
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HIV-1 is characterized by high genetic heterogeneity which is a challenge for developing therapeutics
it is necessary to understand the extent of genetic variations that HIV is undergoing in North India
The objective of this study was to determine the role of genetic and functional role of Vif on APOBEC3G degradation
Vif is an accessory protein involved in counteracting APOBEC3/F proteins
Genetic analysis of Vif variants revealed that Vif C variants were closely related to South African Vif C whereas Vif B variants and Vif B/C showed distinct geographic locations
This is the first report to show the emergence of Vif B/C in our population
motifs and phosphorylation sites were well conserved
Vif C variants differed in APOBEC3G degradation from Vif B variants
Vif B/C revealed similar levels of APOBEC3G degradation to Vif C confirming the presence of genetic determinants in C-terminal region
High genetic diversity was observed in Vif variants which may cause the emergence of more complex and divergent strains
These results reveal the genetic determinants of Vif in mediating APOBEC3G degradation and highlight the genetic information for the development of anti-viral drugs against HIV
Importance: Vif is an accessory HIV-1 protein which plays significant role in the degradation of human DNA-editing factor APOBEC3G
thereby impeding the antiretroviral activity of APOBEC3G
It is known that certain natural polymorphisms in Vif could degrade APOBEC3G relatively higher rate
This is the first report from North India showcasing genetic variations and novel polymorphisms in Vif gene
but for the first time we observed putative B/C recombinants with a little high ability to degrade APOBEC3G indicating adaptation and evolving nature of virus in our population
Indian Vif C variants were able to degrade APOBEC3G well in comparison to Vif B variants
These genetic changes were most likely selected during adaptation of HIV to our population
These results elucidate that the genetic determinants of Vif and highlights the potential targets for therapeutics
there is no genetic information available for Vif gene which warrant for genetic study of Vif in our population
we genetically characterized Vif gene from HIV-1 infected individuals of North India
This study revealed the co-circulation of Vif B and Vif C subtypes which might create a condition conducive for the generation of Vif B/C recombinants
The possible sequence similarity of our Vif variants with the global Vif variants were analysed by constructing phylogenetic tree
Various natural substitutions were observed which may have conferred high pathogenicity advantage to virus
Vif C variants resulted in little high APOBEC3G degradation in comparison to Vif B variants whereas Vif B/C recombinants were similar to Vif C in causing APOBEC3G degradation
This study discusses the influence of genetic variations of Vif on APOBEC3G degradation
(A) Phylogenetic tree of unique Vif variants with M (A to K including A1
(B) Phylogenetic tree of unique Vif variants with global subtype Vif B and Vif C reference sequences
Each reference sequence was labelled with subtype
followed by the country of isolation and accession number
filled circles represent C variants and filled rectangles represent B/C recombinants
1,000 replicates) was indicated with an asterisk (*) at the corresponding nodes of the tree and the scale bar represents the selection distance of 0.01 nucleotides per position in the sequence
(A) VifD64 is a representative of Vif B variant. (B) VifS1 is a representative of Vif C variant. (C) VifA6 is a representative of Vif B/C recombinant. Bootscan analyses were performed using consensus B, C and D shown in blue, red and green coloured arrows respectively with variants.
</a>Multiple sequence alignment of Vif variants
Multiple sequence alignment for the predicted amino acid sequences of unique Vif variants with consensus Vif C and Vif B
amino acids identical to Vif C and Vif B are denoted by asterisk (*)
Polymorphisms are denoted in alphabetical letters
Predicted major functional domains are represented at the top of the sequences within the boxes
R19N change was observed in F1 box of about 76%; S23R
V98I and D99E changes were observed in RNA binding region of about 76%
43% and 43% respectively; R63K change was observed in FG box of about 76%; HCCH region showed A123T and H127R in 53% of variants; K158Q was observed in multimerization region in 53% of variants; SOCS region showed V166I and T167K in 43% and 36% of variants respectively; K176N and T180I were observed in G box in 36% of variants
These regions were conserved but a change in R41G was found in 25% of variants
G37D and H48N were observed in 75% of variants which may modulate APOBEC3G/F degradation
The amino acid region (from 85 to 99 and 169 to192) is shown to mediate Vif-APOBEC3G binding; we observed S95N
D99E and K176N changes in our population which might alter the viral infectivity
</a>Amino acid sequence pattern of Vif variants
(A) Amino acid signature pattern of Vif variants with consensus Vif B
(B) Amino acid signature pattern of Vif variants with consensus Vif C
The X-axis represent the amino acid consensus sequence of Vif B and C with the functional domains viz
Trp-rich (N-terminal Tryptophan rich region from 1–21aa)
F1-box (Interaction with host APOBEC3F from 14–17aa)
RNA (RNA binding from 1–71aa and 75–114aa)
G-box (Interaction with host from 40–44aa and 170–192aa)
FG-box (Interaction with host APOBEC3F and from 54–72aa)
F2-box (Interaction with host APOBEC3F from 74–79aa)
HCCH motif (zinc-binding domain from 108–139aa)
BC motif (Interaction with host elongin BC complex from 144–153aa)
SOCS-box (Interaction with host SOCS from 139–176aa)
MT (Multimerization from 151–164aa) and the Y-axis represent amino acid frequency observed in variants
</a>Motifs and phosphorylation sites in Vif variants
Vif variants show protein kinase C (PKC) sites (20–22aa
bipartite nuclear localization signal profile (NLS) sites (22–36aa and 167–184aa)
N-myristoylation (NM) sites (71–76aa and 82–87aa)
cAMP and cGMP dependent protein kinase (AGPK) sites (92–95aa and 167–170aa)
amidation (AMD) site (181–184aa) and N-glycosylation (NG) site (186–189aa)
The phosphorylation sites (serine and threonine residues) were marked with asterisk (*) at the corresponding amino acid sites on the top of the consensus C sequence
despite high genetic variations and B/C recombination
(A) APOBEC3G protein expression induced by Vif variants
HEK 293T cells were co-transfected with Vif variants and APOBEC3G-HA
cells were lysed and run on SDS-PAGE and then APOBEC3G protein was checked by immmunobloting with anti-HA antibody
The relative protein intensity of APOBEC3G degradation by Vif variants were measured by subtracting APOBEC3G-HA positive control
Densitometry represents the APOBEC3G degradation by Vif variants
The error bar represents the standard deviation of APOBEC3G degradation and the p-value &lt; 0.05 represents statistical significance of relative intensity of APOBEC3G with the relative intensity of respective Vif (* denote significant difference)
This study shows the genetic architecture of HIV-1 Vif comprised of B and C subtypes
This co-circulation of subtypes within a specific population led to the emergence of novel B/C recombinants in the ORF of Vif among North Indians which was observed to be statistically significant (**p &lt; 0.05) i.e
the proportion of recombinants have increased marginally (~3 to 5%) in the last few years implying the stable emergences of recombinants indicating the evolving tendency of the virus in our country
Viral genetic recombination analysis also confirmed the existence of B/C recombinants with precise breakpoint in Vif gene
These recombinants may contribute for generation of more complex viruses in our population; therefore it is essential to understand the genetic makeup of circulating recombinants and their prevalence in North India which will help in designing effective therapeutics against various circulating strains
Phylogenetic analysis of Vif C variants showed sequence similarity with South African C variants whereas Vif B variants showed sequence similarity with the subtype B from different geographic regions that have resulted from multiple introductions of HIV-1 strains from China and other countries viz
probably due to itinerant travellers from different countries
Our data elucidates the importance of phylogenetic and recombination analyses in determining the sequence similarity between our variants with the highly prevalent global strains of HIV-1 and contribute to the better understanding of virus with respect to natural selection and adaption of viral strains
Polymorphism analysis showed certain conserved substitutions which may favour virus for its optimal activities in causing pathogenicity in host cells
these substitutions may involve in down-regulating or enhancing the functional activities of Vif and might also aid in the generation of more complex and divergent strains
targeting the natural substitutions using RNAi mechanism may help in modulating HIV-1 infection
It is important to note that this study is an attempt to analyze Vif variants from ART naïve and ART receiving patients of North India
in relation to the contribution of this trait to pathogenicity
viral evolution and possible implications for ART regimen and vaccine design
low genetic diversity and lesser recombinants were observed among HIV-1 infected individuals receiving ART who showed improved CD4 counts and decreased viral loads implying the importance of ART in restoring immunity
Domain conservation analysis based on sequence profiles of 105 HIV-1 infected patients from North India revealed the conservation at various functional domains viz
ElonginC and SOCS binding domains highlighting the target sites for designing Anti-viral drugs
Motif sites analysis showed conservation at various motifs in variants; remarkably identified nine phosphorylation sites were conserved
The dN/dS analysis suggests purifying selection implying genetic integrity of Vif to maintain the functional activity of Vif
It is necessary to have a matrix of variants in HIV-1 affected population since it will have a predictive value in determining the type of Anti-viral drug targets
Based on sequence analysis of Vif variants in a specific population
it is possible to map cytotoxic T lymphocyte (CTL) epitopes and enhance immune response of HIV-1 infected individuals against virus
To explore the relationship between Vif-mediated APOBEC3G degradation
dual transfection with Vif variants and APOBEC3G was carried out in HEK 293T cells
This assay clearly showed that Vif B variants (Vif D43
D48 and E48) degrades APOBEC3G whereas Vif B/C recombinants (VT3 and VT4) carrying the N terminal region of Vif B and the C terminal of Vif C resulted in a little high APOBEC3G degradation as compared to Vif B and similar levels to Vif C confirming the vital role of C-terminal region in inducing APOBEC3G degradation
This differential potential of Vif variants was due to the genetic variations in Vif B
This study on Vif from HIV-1 infected individuals (n = 105) revealed Indian Vif sequences (VifS1
These matrixes of variants can be used in a combination for the understanding of Vif-host relationship and to develop targets against HIV in our population
This study deciphers that HIV-1 virus continue to evolve in the local population
but current pace of evolution is not so alarming
Whether the dynamics of variation in the evolving population of viruses will retain its current pace or will it be altered is difficult to predict
Whether host immune response is adequate to contain the variations in the evolving virus and whether other factors (host immune response
etc) are likely to influence it remains to be seen
Coinfections such as Tuberculosis may have profound impact on the host immune response thereby compelling the virus to evolve new strategies to synergize with Mycobacterium tuberculosis
in the genetic and functional characteristics of the virus which may be subtle or gross for finding a better-fit to the situation in the host encountered by the virus
periodic analysis of the virus for selection pressure-dictated variants becomes necessary to determine the trend of viral evolution
if the variants are likely to pose a public health problem
if changes are necessary in the modality of ART
if newer viral targets can be obtained for prophylactic or therapeutic intervention and finally
if the biology of the virus is underpinning a new element in basic sciences
Our data indicates the genetic and functional role of Vif in causing APOBEC3G degradation
targeting Vif by utilizing novel RNAi technology will help in perturbing the viral infectivity
This study revealed various natural genetic determinants of Vif in mediating the functional activity of Vif in our population
thus this study will immensely help in designing the potential targets based on Vif-APOBEC3G relationship against HIV/AIDS
our data demonstrates that HIV-1 virus is evolving relevant to adaptation among North Indians through its ability to generate advantageous substitutions and recombinants in Vif gene to enhance its specific functional activities in the host cells
HIV-1 infected patients (n = 105) were collected from North India who were registered and monitored at the immunodeficiency clinics of Guru Teg Bahadur (GTB) hospital
Delhi and Post Graduate Institute of Medical Education and Research (PGIMER)
Chandigarh during the period from 2004 to 2010
Number of males (45%) and females (38%) were chosen for this study along with vertical transmission variants (17%)
This study was approved by Research Project Advisory Committee
Institutional Biosafety Committee and Institutional Ethical Committee from Human research of University College of Medical Sciences and Guru Teg Bahadur Hospital
India and from Post Graduate Institute of Medical Education and Research
These institutes are mentored by National AIDS Control Organization (NACO)
Government of India that provides free of cost ART to HIV-1 seropositive patients under a structured HIV/AIDS Control Programme
These ethics committees approved the written informed consent which obtained from HIV-1 infected patients and from guardians of HIV-1 infected children participants involved in this study
The methods were carried out in “accordance” with the approved guidelines
Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of HIV-1 infected individuals using Trizol reagent (Invitrogen)
PBMCs were washed with PBS and 1 ml of Trizol was added to 50–100 mg cells
200 μl of chloroform was added and mixed by vortexing
This was incubated for 5 minutes at 37 °C and centrifuged at 13,200 rpm for 15 minutes
The upper aqueous phase was transferred to the fresh tube and 500 μl of isopropanol was added to precipitate RNA
The mix was incubated at 37 °C for 10 minutes and centrifuged at 13,200 rpm for 10 minutes at 4 °C
The supernatant was discarded and pellet was washed with 75% ethanol
The pellet was air dried and dissolved in nuclease free water by heating at 55°–60 °C for 10 minutes
This RNA was reverse transcribed to form complementary DNA (cDNA) using ImProm-IITM Transcription system (Promega)
1 μg of RNA was reverse transcribed into cDNA using 20 pmol/μl of random primers and incubated at 70 °C for 15 minutes and kept at 4 °C
Reverse transcription mix containing 1X reaction buffer
rRNasin RNase inhibitor and reverse transcriptase was added to it and incubated at 25 °C for 5 minutes
5 μl of cDNA product was used for amplification of Vif using the following primers:
Forward primer: 5′-GCGGATCCATGGAAAACAGATGGCAGG-3′
Reverse primer: 5′-GCCTCGAGCTAGTGTCCATTCATTGTATGG-3′
PCR was carried out in a 15  μl reaction volume
The reaction mixture contained 500 ng genomic DNA (2.0 μl)
0.25  μl of Takara Taq DNA polymerase and 8.88 μl of DNase/RNase free water
PCR conditions for the above primer sets were as follow: Initial denaturation at 94 °C for 5 minutes (1 cycle)
30 cycles of denaturation at 94 °C for 30 seconds
annealing at 65 °C for 45 seconds and extension at 72 °C for 40 seconds and a final extension at 72 °C for 5 minutes (1 cycle)
PCR amplified products were analyzed on 1.5% agarose gel
The gel purified PCR products were cloned in pGEM-T Easy vector system
The ligation reaction was incubated at 4 °C for 10 hours and the ligation mix was then plated on LB ampicillin plates with E.coli DH5α strain as host
The plates were then incubated overnight at 37 °C
The positive clones were selected by picking a single colony and grown in 5 ml LB Broth with ampicillin antibiotic (100 μg/ml) and incubated overnight at 37 °C
Plasmid DNA was isolated from the culture by QIAprep Spin Mini Kit
The positive clones were screened by restriction digestion of plasmid DNA with EcoRI in a 10 μl reaction volume at 37 °C for 2 hours
The digested products were analyzed on 1.5% agarose gel after electrophoresis and the amplified bands were screened for positive clones after restriction digestion
Five positive clones from each individual were sequenced from LabIndia and SciGenom laboratories by dideoxy chain termination method
The cloning and sequencing were carried out twice for the positive clones to avoid PCR generated errors
A known HIV-1 NL4-3 sequence was included for PCR amplification as a control to assess errors generated by Takara Taq polymerase
The reliability of node was tested using the bootstrap method with 1000 replicates
The phylogenetic trees were constructed for Vif representative variants (12 Vif B variants
3 Vif C variants and 3 Vif B/C recombinants) from North India and reference sequences which were retrieved from HIV Sequence database that includes subtype B and C sequences obtained from different parts of the world include America
The basic criterion for choosing these reference sequences is that to cover all regions in worldwide for analysis in order to determine the sequence similarity with global reference sequences
The nucleotide sequences of Vif were translated into amino acid sequences by GeneRunner
The multiple sequence alignments were made for all variants with consensus B and C using ClustalW 2.1
It is already well known that subtype C is highly prevalent in India followed by subtype B and choosing consensus B and C sequences for analysis purpose is the idea behind the subtype prevalence in India and were retrieved from HIV Sequence database
Novel polymorphisms were identified and their allele frequencies were calculated from total variants (n = 105)
The amino acid sequence conservation was determined from the multiple sequence alignments of variants with the corresponding consensus B and C sequences using sequence alignment tool in MEGA5 software
HEK-293T (Human Embryonic Kidney 293 cells; NIH AIDS Reagent Programme) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and 100 units penicillin
0.1 mg streptomycin and 0.25 μg amphotericin B per ml at 37 °C in the presence of 5% CO2
All transfections were performed using Lipofectamine 2000 (Invitrogen) reagent
anti- HA tag polyclonal antibody (Clontech)
anti-GAPDH antibody (Cell Signalling Technology)
anti-vif monoclonal antibody (NIH AIDS Reagent Programme)
anti-Rabbit IgG conjugated to HRP (Jackson Immunoresearch)
anti-Mouse IgG conjugated to HRP (Jackson Immunoresearch) were used in the experiments
Evidence for HTLV-III infection in prostitutes in Tamil Nadu (India)
Determination of the rate of base-pair substitution and insertion mutations in retrovirus replication
High rate of recombination throughout the human immunodeficiency virus type 1 genome
Adaptive evolution of human immunodeficiency virus-type 1 during the natural course of infection
A new human immunodeficiency virus derived from gorillas
HIV Databases. &amp;lt;<a href="http://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="http://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html">www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html</a>&amp;gt;
Human immunodeficiency virus type 1 subtype distribution in the worldwide epidemic: pathogenetic and therapeutic implications
Dynamic correlation between intrahost HIV-1 quasispecies evolution and disease progression
APOBEC3G encapsidation into HIV-1 virions: which RNA is it
Natural variation in Vif: differential impact on APOBEC3G/3F and a potential role in HIV-1 diversification
Human immunodeficiency virus superinfection and recombination: current state of knowledge and potential clinical consequences
Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 34
Recombination increases human immunodeficiency virus fitness
The role of recombination in the emergence of a complex and dynamic HIV epidemic
Genetic characterization of natural variants of Vpu from HIV-1 infected individuals from Northern India and their impact on virus release and cell death
Tat-Vpr interaction and cell apoptosis by natural variants of HIV-1 Tat exon 1 and Vpr from Northern India
Genetic architecture of HIV-1 genes circulating in north India &amp; their functional implications
The Indian journal of medical research 134
Molecular and genetic characterization of natural HIV-1 Tat Exon-1 variants from North India and their functional implications
A web-based genotyping resource for viral sequences
Differential requirement for conserved tryptophans in human immunodeficiency virus type 1 Vif for the selective suppression of APOBEC3G and APOBEC3F
Human immunodeficiency virus type 1 Vif protein is an integral component of an mRNP complex of viral RNA and could be involved in the viral RNA folding and packaging process
Advances in the structural understanding of Vif proteins
Nuclear localization of HIV type 1 Vif isolated from a long-term asymptomatic individual and potential role in virus attenuation
Assembly of HIV-1 Vif-Cul5 E3 ubiquitin ligase through a novel zinc-binding domain-stabilized hydrophobic interface in Vif
The SOCS box of suppressor of cytokine signaling-1 is important for inhibition of cytokine action in vivo
Proceedings of the National Academy of Sciences of the United States of America 98
Potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type 1 (HIV-1) Vif proteins
HIV-1 infection requires a functional integrase NLS
C-terminal half of HIV-1 Vif C possesses major determinant for APOBEC3G degradation
Ubiquitination of APOBEC3G by an HIV-1 Vif-Cullin5-Elongin B-Elongin C complex is essential for Vif function
High replication fitness and transmission efficiency of HIV-1 subtype C from India: Implications for subtype C predominance
Proceedings of the National Academy of Sciences of the United States of America 106
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting
position-specific gap penalties and weight matrix choice
Prospects for inferring very large phylogenies by using the neighbor-joining method
Proceedings of the National Academy of Sciences of the United States of America 101
HIV Database Tools. &amp;lt;<a href="http://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="http://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html">http://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html</a>&amp;gt;
Disease relevance of T11TS-induced T-cell signal transduction through the CD2-mediated calcineurin-NFAT pathway: Perspectives in glioma immunotherapy
Mechanism of apoptotic induction in human breast cancer cell
by an analog of curcumin in comparison with curcumin--an in vitro and in silico approach
angiogenesis and metastasis in breast cancer cells via NF-kappaB pathway-A comparative study with curcumin
Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie 74
Apoptosis induction by an analog of curcumin (BDMC-A) in human laryngeal carcinoma cells through intrinsic and extrinsic pathways
<a data-track="click" data-track-action="download citation references" data-track-label="link" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/srep15438?format=refman&amp;flavour=references">Download references</a>
India for helping in the collection of HIV-1 infected blood samples
Source of Funding: This study was supported by Department of Biotechnology (BT/PR10599/Med/29/76/2008) and Indian Council of Medical Research (HIV/50/142/9/2011-ECD-II)
Present address: Division of Infectious Diseases
University College of Medical Sciences &amp; Guru Teg Bahadur Hospital
Vaishali Panwar &amp; Vishnampettai G Ramachandran
Department of Biochemistry and Molecular Biology
Kumaravel Mohankumar &amp; Subhashree Sridharan
Jawaharlal Institute of Postgraduate Medical Education and Research
Conceived and designed the experiments: L.R.
Contributed reagents/materials/analysis tools: L.R.
<a data-test="citation-link" data-track="click" data-track-action="download article citation" data-track-label="link" data-track-external="" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/srep15438?format=refman&amp;flavour=citation">Download citation</a>
CHAPEL HILL, NC (PRWEB) January 10, 2017 -- VIF International Education, an education partner of K-12 schools and districts, announced today that it is changing its name to <a href="http://participate.com" title="Participate" target="_blank" rel="nofollow">Participate</a>
The change reflects the organization’s broader commitment to educator development and its expertise in providing continuous learning opportunities that empower teachers and inspire students to be active contributors to their communities and our world
our growing national and international work now includes instructional design
program management and education technology
In May of 2016, <a href="http://www.educationnews.org/online-schools/vif-international-education-acquires-participate-learning/" title="VIF International Education acquired Participate Learning" target="_blank" rel="nofollow">VIF International Education acquired Participate Learning</a>
a leading online platform for educational resource discovery and collaboration
The acquisition positioned the organization to deliver best-in-class technology to enhance and expand professional learning and collaboration opportunities for educators
It represented what became the preeminent U.S
cultural exchange program for teachers and afforded us the opportunity to pursue other pressing education needs,” said Participate CEO David Young
we believe Participate more accurately reflects these broader education pursuits and the enhanced technology capabilities that will help us reach more students and teachers than we ever thought possible - it recognizes our past
present and future in support of collaborative
experiential and global learning for all.”
Visiting International Faculty (VIF) was founded in 1987 as a family business that sought to promote the value of international perspectives in education by providing universities with international faculty recruitment
The organization shifted focus to K-12 education in 1989 when state departments of education began pursuing world language learning for K-12 students
VIF has placed thousands of international teachers in K-12 classrooms across the United States as a designated U.S
Department of State Exchange Visitor Program sponsor
The organization continues to be committed to cultural exchange and supports international teachers’ success in the classroom as it firmly believes that exposing students to intercultural experiences at the K-12 level is key to building global competence - a critical skill for success in our increasingly interconnected world
the organization also provides schools and districts with leading-edge technology
comprehensive frameworks and support services to impact student outcomes by improving teacher practice
Many of its partners are distinguished by school-wide commitments to strengthen academic achievement for diverse student populations through immersive language learning opportunities and equitable access to global
The new name is effective immediately and will be implemented across the organization's products and services throughout the remainder of 2017
About Participate Participate (<a href="http://participate.com" title="http://www.participate.com" target="_blank" rel="nofollow">http://www.participate.com</a>) partners with schools and districts to provide leading-edge technology
comprehensive frameworks and support services
These programs and services impact student outcomes by improving teacher practice through collaborative professional learning
educators have used Participate’s professional development and curriculum
language acquisition and cultural exchange teacher programs to create engaging learning environments that empower teachers and inspire students to create impact on a global scale
Participate is a certified B Corp and ‘Best for the World’ honoree headquartered in Chapel Hill
TJ Scholl, Participate, https://www.participate.com/, 919-265-5065, <a href="/cdn-cgi/l/email-protection#7307195d00101b1c1f1f33031201071a101a031207165d101c1e" rel="nofollow" target="_blank">[email&#160;protected]</a>
Do not sell or share my personal information:
<a href="https://aws.amazon.com/directconnect">AWS Direct Connect</a> (DX) <a href="https://aws.amazon.com/about-aws/whats-new/2020/05/enhanced-monitoring-capabilities-for-aws-direct-connect/">recently launched</a> support for virtual interface (VIF) metrics in <a href="https://aws.amazon.com/cloudwatch">Amazon CloudWatch</a>
CloudWatch can now track metrics at the DX VIF level and provide greater insight into utilization
You can set up alarms based on metrics and trigger actions to remediate problems
I’ve heard from many customers that they wanted greater visibility into traffic utilization when using multiple VIFs on the same connection – dedicated or hosted
I’m excited about this release as there is now a solution
I dig into this new functionality and how it compares to the prior capability
Before this launch it was possible to see aggregated metrics at the Direct Connect connection
it was not possible to view individual VIFs and determine throughput utilization
a 10-Gbps DX connection has a transit VIF for AWS Transit Gateway connectivity
a public VIF for connectivity to public AWS resources
and a private VIF for connectivity to a VMware environment
Looking at the screenshot you can see that there was a bit of a spike in throughput
there is not an easy way to drill down and determine which VIF is the source of this traffic
Now that that there are VIF level metrics we see a new set of metrics in the CloudWatch console
I can now dig in further to see which VIF is responsible for the traffic
Here I can see that there are metrics for the VIF that resides within my DX account
you can see that the peak here is only around 8-Mbps
It turns out that the other VIFs are hosted VIFs and actually reside in a different account than the DX connection
Fortunately it is quite simple to share CloudWatch metrics across accounts
CloudWatch metrics can be viewed <a href="https://docs.aws.amazon.com/AmazonCloudWatch/latest/monitoring/Cross-Account-Cross-Region.html">cross-region and cross-account</a>
I can see a drop-down in the console to view data from the shared account
I can select an account and view its metrics
I can graph these metrics alongside the VIF that exists within the DX account
Note that there is a fee if anomaly detection is enabled, so be sure to check the CloudWatch <a href="https://aws.amazon.com/cloudwatch/pricing/">pricing page</a>
I enabled this on a VIF metric for BpsEgress (bytes per second egress) as shown in the following image
 Once anomaly detection is enabled I can see that traffic spikes are outside the ordinary baseline for traffic
I also can see the expected rate of traffic
In this post I showed how VIF CloudWatch metrics can be used and shared between accounts. For a full list of CloudWatch metrics supported by DX you can view the <a href="https://docs.aws.amazon.com/directconnect/latest/UserGuide/monitoring-cloudwatch.html">documentation</a>
With this new capability even greater visibility into DX utilization is now possible
Volume 13 - 2022 | <a class="ArticleLayoutHeader__info__doi" href="https://doi.org/10.3389/fmicb.2022.828430">https://doi.org/10.3389/fmicb.2022.828430</a>
Human immunodeficiency virus type 1 (HIV-1) has RNA genome and depends on host cellular machinery for most of its activities
Host cellular proteins modulate the expression and activity of viral proteins to combat the virus
HIV-1 proteins are known to regulate each other for the benefit of virus by exploiting these modulations
we report that HIV-1 Vif increases the levels of Tat via AKT signaling pathway
We show that HIV-1 Vif activates AKT signaling pathway by inducing phosphorylation of AKT
increases the levels of Tat protein in ubiquitin-dependent manner by inducing Ubiquitin Specific Protease 17 (USP17)
which is a deubiquitinase and stabilizes Tat protein
HIV-1 proteins exploit AKT signaling pathway to promote viral replication
so we investigated the effect of Vif on the expression of Tat to find out how HIV-1 is benefited by activating AKT signaling pathway via its two proteins
We found that Vif increased the levels of Tat protein
Vif was also found to increase the LTR transcription mediated by Tat protein
Inhibition of AKT phosphorylation abrogated Vif-mediated increase in levels of Tat protein
Mdm2 (target of AKT) was found to increase the levels of Tat via a deubiquitinase
AKT signaling pathway was playing an important role in the regulation of HIV-1 Tat by Vif via Mdm2 mediated stabilization of USP17
This study can have significant implications toward better understanding of the several mechanisms of HIV-1&#x2013;mediated exploitation of host machinery and viral pathogenesis
Human embryonic kidney 293T (HEK-293T) and Tzm-Bl cells were maintained in Dulbecco modified eagle medium (Himedia Laboratories
India) supplemented with 10% fetal bovine serum (Gibco
and 0.25 &#x03BC;g amphotericin B per ml at 37&#x00B0;C in the presence of 5% CO2 in a humidified incubator
and U1 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (Gibco
and 0.25 &#x03BC;g amphotericin B (Himedia Laboratories
India) per milliliter at 37&#x00B0;C in the presence of 5% CO2 in a humidified incubator
Transfections were performed using Lipofectamine 2000 (Invitrogen
United States) reagents using the manufacturer&#x2019;s protocol
Plasmid Myc Vif was made by cloning pNL4-3&#x2013;derived gene vif in pCMV-Myc plasmid from Clontech, United States, as described earlier (<a href="#B2">Arora et al., 2014</a>)
pBlue3&#x2032;LTR-luc was obtained from NIH AIDS Reference and Reagent Program of NIH
Glutathione S-transferase (GST) Tat was generated by cloning pNL4-3 derived tat gene in pGEX-4T1 vector from Addgene
HA Tat and Flag Tat were purchased from Addgene
HA Mdm2 was purchased from Sino Biologicals
and HA Myr AKT were kind gifts from Hui Kuan Lin
Renilla luciferase plasmid was a kind gift from Vivek Natrajan
His Ub plasmid was gifted by Dimitris Xirodimas
Human embryonic kidney 293T cells were transfected with gene of interest for 24 h
The cells were harvested and lysed in RIPA lysis buffer (1% NP-40
Protein estimation was carried out using BCA Protein Assay Kit (Pierce
An equal amount of protein was loaded on sodium dodecyl sulfate&#x2013;polyacrylamide gel electrophoresis (SDS-PAGE) and was transferred to nitrocellulose membrane
The membranes were blocked with 5% non-fat dry milk (Himedia Laboratories
anti&#x2013;phospho-AKT (S473) (Cell Signaling Technology)
The secondary antibodies used were anti-rabbit/mouse&#x2013;horseradish peroxidase&#x2013;conjugated (Jackson ImmunoResearch)
Blots were developed using ECL (enhanced chemiluminescence) reagent
To study the degradation kinetics of proteins
HEK-293T cells were transfected with gene of interest for 24 h and treated with CHX (100 &#x03BC;g/mL; Sigma)
Cell lysates were prepared at indicated time points and subjected to 10% SDS-PAGE followed by Western blot analysis as described above
In vivo ubiquitination assay was performed to detect ubiquitylated proteins in transfected mammalian cells
HEK-293T cells were cotransfected with plasmid encoding desired gene and His-Ub (6 &#x00D7; histidine&#x2013;ubiquitin) for 24 h
20 &#x03BC;M MG132 (Sigma-Aldrich) was added
and the cells were further incubated for 8 h
The cells were lysed in buffer A (6 M guanidinium-HCl
Ni-NTA beads were added to the supernatant
and the mixture was incubated at room temperature for 6 h while rotating
the beads were washed with buffer A and buffer TI (25 mM Tris
The ubiquitinated proteins were eluted in buffer containing 200 mM imidazole
The eluates were resolved by SDS-PAGE followed by Western blot analysis
pGEX-4T1 vector containing the desired gene was transformed in BL21 strain of Escherichia coli for expression and subsequent purification
The bacterial culture was induced with 0.5 mM IPTG at 16&#x00B0;C for 16 h
The cells were lysed by adding lysozyme (1 mg/mL) at 4&#x00B0;C with gentle shaking
DTT was added to the bacterial lysate after lysozyme treatment (100 &#x03BC;L of 1 M DTT)
This was followed by sonication and extraction of proteins with Triton X-100
The solution was centrifuged at 12,000 revolutions/min (rpm) for 15 min at 4&#x00B0;C
The supernatant was then used for binding to glutathione beads at 4&#x00B0;C for 3 h
The beads were centrifuged at 2,500 rpm for 2 min at 4&#x00B0;C
The beads were washed until the supernatant stopped giving color with Bradford reagent
GST alone and GST-tagged proteins were expressed and purified as described previously
HEK-293T cells were transfected with gene of interest for 36 h
Ten micrograms of GST-tagged protein was incubated with the cell lysate at 4&#x00B0;C for 4 h
and the beads were washed five to six times with chilled 1 &#x00D7; phosphate-buffered saline (PBS)
The beads were boiled in Laemmli buffer and subjected to SDS-PAGE followed by immunoblotting with anti-Myc and anti-GST antibodies
The protein&#x2013;protein interaction was studied by coimmunoprecipitation
The genes of interest were cotransfected in HEK-293T cells for 24 h
For affinity tag-based immunoprecipitation
cell lysates were prepared in CelLytic M and cell lysis reagent (Sigma) and were incubated with anti-Myc agarose beads (Sigma) at 4&#x00B0;C overnight
The beads were washed with IP buffer (Sigma)
The purified protein complex bound to anti-Myc agarose beads was resolved by SDS-PAGE and subjected to Western blot analysis
Pierce&#x2122; Direct IP kit (Thermo Scientific) was used to pull down a protein without tag
HEK-293T cells were transfected with desired plasmids
and antibody-conjugated agarose resin was added
resin was pelleted and washed with wash buffer
The immunoprecipitated proteins were eluted using elution buffer
The aqueous solution containing eluted proteins was boiled with SDS&#x2013;PAGE loading buffer for 5 min and analyzed by Western blotting
Luciferase reporter assay was performed using the dual-luciferase reporter assay kit (Promega
HEK-293T cells were cotransfected with luciferase reporter plasmid and the plasmids encoding genes of interest
Renilla luciferase was used as control to normalize the transfection efficiency
Empty pcDNA3.1 vector was used to equalize the amount of DNA transfected in each well
cell were harvested and lysed in lysis buffer (Promega
Luciferase activity was measured by luminometer (Tecan
Switzerland) using two substrates (Promega
United States): one for firefly luciferase and another for Renilla luciferase (mixed with Stop and Glo buffer)
The readings of firefly luciferase activity were normalized with those of Renilla luciferase activity to get the true luciferase reporter activity
All the experiments were repeated three to four times
Results obtained are presented as mean &#x00B1; standard error of the mean (s.e.m)
p-values were calculated by a two-tailed t-test
Only values with p &#x003C; 0.05 were considered significant
Human immunodeficiency virus type 1 Vif induces phosphorylation of AKT at Ser473
(A) HEK-293T cells were cotransfected with HA AKT
Cell lysates were analyzed by Western blotting with anti-HA
(B) HEK-293T cells were transfected with increasing amounts of Myc Vif expression plasmid for 24 h
Cell lysates were analyzed by Western blotting with anti-AKT
(C) HEK-293T cells were transfected with Myc Vif expression plasmid and treated with AKTi (5 &#x03BC;M) as indicated for 24 h
(D) HEK-293T cells were transfected with Myc Vif and treated with increasing dose of AKTi (3 and 5 &#x03BC;M) for 24 h
(E) HEK-293T cells were treated with AKTi (5 &#x03BC;M) and transfected with increasing amount of Myc Vif (2 and 4 &#x03BC;g) for 24 h
(F) HEK-293T cells were transfected with AKT siRNA and Myc Vif
Densitometry analysis was performed using ImageJ and shown as bar graph with error bars
Data obtained are presented as mean &#x00B1; s.e.m
p-value was calculated by two-tailed t-test [&#x002A;p &#x003C; 0.05
suggesting Vif-mediated increase in the levels of Tat protein
Human immunodeficiency virus type 1 Vif increases the expression of Tat via AKT signaling pathway
(A) HEK-293T cells were cotransfected with HA Tat and Myc Vif plasmids as indicated for 24 h
Cell lysates were analyzed by Western blotting using anti-HA
(B) HEK-293 T cells were transfected with HA Tat either alone or along with Myc Vif for 24 h
Cells were treated with cycloheximide (100 &#x03BC;g/mL) for the indicated time periods
Cell lysates were subjected to Western blot analysis using anti-HA
Densitometric analysis was done by ImageJ and shown as line graph with error bars
(C) HEK-293T cells were cotransfected with HA Tat and Myc Nef plasmids as indicated for 24 h
(D) HEK-293T cells were cotransfected with HIV-1 LTR-luc reporter
and Myc Vif expression plasmids as indicated for 24 h
and dual luciferase reporter assay was performed using luminometer
Relative luciferase activity is shown as bar graph
(E) GST Tat and GST alone bound with GST beads were incubated with in vitro synthesized Vif for 2 h at 4&#x00B0;C
Vif specific antiserum was used to probe the Vif protein in Western blot analysis
(F) Tzm-Bl cells were transfected with HA Tat and Myc Vif plasmids for 24 h
Cell lysates were subjected to immunoprecipitation using anti-HA agarose beads at 4&#x00B0;C overnight
beads were washed with IP buffer and boiled with PAGE loading buffer followed by SDS-PAGE and Western blotting using anti-Myc antibody
(G) HEK-293T cells were cotransfected with HA Tat and Myc Vif plasmids as indicated and treated with AKTi (5 &#x03BC;M) for 24 h
(H) HIV-1 replication in U1 cells was induced by PMA (100 &#x03BC;g/mL)
and cells were treated with AKTi (5 &#x03BC;M) for 24 h
Cell lysates were analyzed by Western blotting using anti-p24
These results suggest intracellular interaction of Tat and Vif proteins
we concluded that total ubiquitination of Tat is reduced in the presence of Mdm2 despite the fact that Mdm2 induces K63 ubiquitination of Tat
This might be the reason that the difference between the ubiquitination levels of Tat in the presence or absence of Mdm2 is less than expected
These results indicate that Mdm2 is the candidate protein of AKT signaling pathway
which increases the levels of Tat in ubiquitin-dependent manner
Mdm2 stabilizes the expression of HIV-1 Tat in ubiquitin-dependent manner
(A) HEK-293T cells were transfected with HA Tat either alone or along with HA Mdm2 for 24 h
Cell lysates were subjected to Western blot analysis using anti-HA and anti-GAPDH antibodies
(B) HEK-293 T cells were transfected with HA Tat either alone or along with HA Mdm2 for 24 h
(C) HEK-293T cells were transfected with Mdm2 siRNA and HA Tat for 48 h
(D) HEK-293T cells were cotransfected with His Ub
Cells were treated with MG132 (20 &#x03BC;M) for 8 h
Cell lysates were subjected to immunoprecipitation with Ni-NTA beads followed by Western blotting with anti-Flag antibody
p-value was calculated by two-tailed t-test (&#x002A;p &#x003C; 0.05
These results indicate that USP17 stabilizes HIV-1 Tat by inducing its deubiquitination
Ubiquitin Specific Protease 17 stabilizes the expression of Tat by deubiquitination
(A) HEK-293T cells were cotransfected with HA Tat and His USP17 plasmids for 24 h
(B) HEK-293T cells were transfected with HA Tat either alone or along with His USP17 for 24 h
(C) HEK-293T cells were cotransfected with His Ub
Cell lysates were subjected to immunoprecipitation with Ni-NTA beads followed by Western blotting with anti-HA antibody
(D) HEK-293T cells were transfected with HA Tat encoding plasmid
cell lysates were subjected to immunoprecipitation using anti&#x2013;USP17 antibody&#x2013;bound agarose beads followed by Western blotting using anti-HA antibody
(E) HEK-293T cells were cotransfected with HA Tat and pNL4.3 for 24 h
Cell lysates were subjected to Western blotting with anti-HA
Mdm2 stabilizes the expression of USP17 in ubiquitin-dependent manner
(A) HEK-293T and Thp-1 cells were treated with AKTi as indicated for 24 h
Cell lysates were subjected to Western blotting using anti-USP17
(B) HEK-293T cells were cotransfected with His USP17 and HA Mdm2 plasmids for 24 h
Cell lysates were analyzed by Western blotting using anti-His
(C) HEK-293T cells were transfected with His USP17 and increasing amounts of HA Mdm2 encoding plasmid
cell lysates were subjected to Western blotting using anti-His
and anti-GAPDH antibodies (D) HEK-293T cells were transfected with increasing amounts of HA Mdm2 plasmid for 24 h
(E) HEK-293T cells were cotransfected with His Ub
Cell lysates were subjected to immunoprecipitation with Ni-NTA beads followed by Western blotting with anti-USP17 antibody
(F) HEK-293T cell lysates were subjected to immunoprecipitation using anti&#x2013;Mdm2 antibody&#x2013;bound agarose beads followed by Western blotting using anti-USP17 antibody
(G) HEK-293 T cells were transfected with siRNA specific to Mdm2
cell lysates were subjected to Western blot analysis using anti-USP17
p-value was calculated by two-tailed t-test (&#x002A;&#x002A;p &#x003C; 0.01)
These results indicate that Mdm2 increases the levels of USP17 in ubiquitin-dependent manner by interacting with it
These results indicate that USP17 is induced by HIV-1 infection
Human immunodeficiency virus type 1 Vif increases the expression of USP17
(A) HEK-293T cells were cotransfected with His USP17 and Myc Vif expression plasmids for 24 h
Cell lysates were subjected to Western blot analysis using anti-His
(B) HEK-293T cells were transfected with pNL4.3 plasmid for 24 h
Cell lysates were analyzed by Western blotting with anti-USP17
(C) U1 and U937 cells were treated with PMA (100 &#x03BC;g/mL) for different time intervals as shown
(D) HEK-293T cells were transfected with pNL4.3 or pNL4.3&#x0394;Vif for 24 h
These results indicate that HIV-1&#x2013;induced enhancement in USP17 protein levels is mediated by Vif
Human immunodeficiency virus type 1 Vif induces proteasomal degradation of Mdm2
(A) HEK-293T cells were cotransfected with HA Mdm2 and Myc Vif expression plasmids for 24 h
HEK-293T cells were transfected with Myc Vif encoding plasmid for 24 h
Cell lysates were analyzed by Western blotting with anti-Mdm2
(B) HEK-293T cells were transfected with HA Mdm2 and Myc Vif expression plasmids for 24 h as indicated and treated with CHX (100 &#x03BC;g/mL) for indicated time periods
and cell lysates were subjected to SDS-PAGE followed by Western blotting using anti-HA
Densitometry analysis was performed using ImageJ and shown as line graph with error bars
(C) HEK-293T cells were cotransfected with HA Mdm2
and Myc Vif encoding plasmids as shown for 24 h
and cell lysates were subjected to SDS-PAGE followed by Western blotting with anti-HA
(D) HEK-293T cells were transfected with Myc Vif expression plasmid and treated with AKTi (5 &#x03BC;M) for 24 h
(E) HEK-293T cells were cotransfected with HA Mdm2 and Myc Vif encoding plasmids for 24 h
(F) HEK-293T cells were cotransfected with His Ub
These results indicate that Vif induces ubiquitin-mediated proteasomal degradation of Mdm2
the effect of Vif on the expression of Tat and the role of AKT signaling pathway in this process were investigated
Vif was found to increase the levels of Tat protein
and Tat-mediated LTR transactivation was also found to be enhanced in the presence of Vif
HIV-1 Vif and Tat were also found to interact with each other
The inhibition of AKT phosphorylation interfered with Vif-mediated increase in Tat levels
was found to induce an increase in the levels of Tat protein
When the mechanism of Mdm2-mediated stabilization of Tat was investigated
it was observed that Mdm2 can up-regulate the levels of HIV-1 Tat protein in ubiquitin-dependent manner via USP17
which stabilizes Tat by deubiquitinating it
HIV-1 also induced an increase in the levels of USP17
Vif-mediated increase in the levels of Tat protein might be independent of Tat-Vif interaction as it has been found to be mediated by AKT signaling pathway
Vif induced proteasomal degradation of Mdm2 protein instead of increasing its expression
which was expected from the inducing effect of Vif on phospho-AKT Ser 473
These results indicate the complex role of AKT signaling pathway in regulation of HIV-1 protein expression and viral replication
we hypothesize that HIV-1 exploits AKT signaling pathway through its activation by Tat and Vif so as to maintain the sufficient levels of Mdm2 in the host cell to increase Tat levels and activity
But when there is more increase in the levels of Mdm2
HIV-1 regulates the expression of Mdm2 via inducing its degradation by Vif so as to prevent Mdm2-mediated proteasomal degradation of Vif
It helps the virus to evade the host restriction machinery more effectively and survive in the host cell
Mechanistic model of HIV-1 Vif&#x2013;mediated up-regulation of Tat via AKT signaling pathway
HIV-1 Tat induces AKT signaling pathway and increases the phosphorylation of AKT and Mdm2
HIV-1 Vif increases the expression of Tat protein via AKT signaling pathway where Mdm2 acts as the mediator
Mdm2 increases the expression of Tat by inducing an increase in the levels of USP17
which stabilizes the levels of Tat by deubiquitination
HIV-1 Vif also induces proteasomal degradation of Mdm2 (blue arrows: previously known phenomena
The original contributions presented in the study are included in the article/supplementary material
further inquiries can be directed to the corresponding authors
SL and VS designed and performed the experiments
This work was supported by Department of Biotechnology and Department of Science and Technology of Government of India
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations
Any product that may be evaluated in this article
or claim that may be made by its manufacturer
is not guaranteed or endorsed by the publisher
Several reagents were obtained from AIDS Reference and Reagent Program of NIH
We thank Hui Kuan Lin (MD Anderson Cancer Center
India) for Renilla luciferase plasmid and Dimitris Xirodimas (University of Dundee) for His Ub plasmid
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Sood V and Banerjea AC (2022) Human Immunodeficiency Virus Type 1 Vif Up-Regulates the Expression of Tat via AKT Signaling Pathway: Role of Ubiquitin Specific Protease 17
Copyright &#x00A9; 2022 Lata, Sood and Banerjea. This is an open-access article distributed under the terms of the <a rel="license" href="http://creativecommons.org/licenses/by/4.0/" target="_blank">Creative Commons Attribution License (CC BY)</a>
*Correspondence: Sneh Lata, <a id="encmail">c25laGxhdGF2aXJvQGdtYWlsLmNvbQ==</a>; Akhil C. Banerjea, <a id="encmail">YWtoaWxAbmlpLmFjLmlu</a>
<a href="/articles/s41598-018-26318-3/metrics" data-track="click" data-track-action="view metrics" data-track-label="link" rel="nofollow">Metrics details</a>
The data from three independent experiments are summarized in the bar graph and represent the mean ± s.e.m (err bar)
The value of controls is arbitrarily set as 100 (%)
Asterisk (*) indicate P &lt; 0.05 and double asterisks (**) indicate P &lt; 0.01 relative to controls (the GraphPad Prism software)
To evaluate the biological activity of the 26 hits, we assessed their effect on hA3G expression in the presence of HIV-1 Vif. 293 T cells were co-transfected with the expression vectors for hA3G-HA and Vif, and then treated with 10 μM of each compound. The results in Fig.&nbsp;<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-018-26318-3#Fig1">1B</a> showed that four compounds
restored hA3G expression in the presence of Vif
compared with that in the cells treated with DMSO
This suggests that the four compounds are able to inhibit the degradation of hA3G by HIV-1 Vif
</a>IMB-301 inhibits the replication of HIV-1 in the presence of hA3G
H9 and SupT1 cells were infected with wild-type HIV-1(NL4-3) in the presence of IMB-293 (A)
The infectivity of nascent viruses in the supernatant was measured by infecting the TZM-bl indicator cells
(E) 293 T cells were co-transfected with pNL4-3Luc(R-E-)
followed by the treatment with various concentration of IMB-301
The supernatants were collected and then used to infect the SupT1 cells
(F) H9 or SupT1cells were incubated with various concentrations of IMB-301 or DMSO for 48 hours
and cytotoxic effects were then measured using the CCK8 assays
Equal volumes of DMSO were added into the culture medium
which contain different concentrations of the compounds
in order to keep constant final DMSO concentration as 1% (v/v)
The value of controls is arbitrarily set as 1 (A–D) or 100% (E and F)
</a>IMB-301 specifically inhibitshA3G but not hA3F degradation. 293 T cells were co-transfected with Vif and eitherhA3G-HA (A) or hA3F-HA (B), followed by the treatment of IMB-301. Then cell lysates were analyzed by WB. Total amounts of DNA were maintained the same between transfections by supplementation of empty vector DNA.
(A) Octet binding of IMB-301 to hA3G was performed as described in Materials and Methods
(B) The predicted binding mode of IMB-301 in the binding site for the homology structure of hA3G-NTD (HM-hA3G-NTD)
(C) The detailed interactions between IMB-301 and hA3G-NTD
we have identified a small molecular inhibitor IMB-301 via virtual screening according to the hA3G model
Further biochemical experiments have shown that IMB-301 binds to hA3G
restores hA3G expression in the presence of Vif
and inhibits the replication of HIV-1 in a hA3G-dependent manner
Our results demonstrate the possibility of inhibiting HIV replication by abrogating the Vif-hA3G interaction with small molecules
293 T and TZM-bl cells were cultured in DMEM (GBICO) supplemented with 10% fetal bovine serum (FBS) (GBICO)
SupT1 cells were maintained in RPMI-1640 (GBICO) containing 10% FBS
H9 cells were maintained in RPMI-1640 (GBICO) containing 10% FBS
Transfections of 293 T cells were performed using Lipofectamine 2000 (Invitrogen) according to the manual from the manufacturer
the supernatants of virus-producing cells were pelleted through a 20% sucrose cushion at 35000 rpm for 60 min (Beckman)
The harvested viral samples were analyzed by western blotting
Western blots were probed with monoclonal antibodies against HIV-1 p24 (NIH)
Detection of proteins was performed by enhanced chemiluminescence (Millipore)
using secondary antibodies anti-mouse (for β-actin) and anti-rabbit (for P24 and HA)
both were purchased from Santa Cruz Biotechnology Inc
Bands in western blots were quantitated using ChemiDoc TMMP (Bio-Rad) automated digitizing system and Image J software
To assess the effect of the four small compounds on HIV-1 infectivity
the experiments were carried out in H9 and SupT1 cells
H9 and SupT1 cells were infected with wild-type HIV-1(NL4-3)
in the presence of the compounds at various concentrations
The infectivity of nascent viruses in the supernatant was measured by infecting the TZM-bl indicator cell for 48 hours (hr)
To assess the effect on the pseudotyped HIV-1
293 T cells were co-transfected with 300 ng pNL4-3Luc(R-E-)
200 ng VSVG and 200 ng hA3G-HA (or pcDNA3.1) plasmid DNA in 6-wellplates
the media was changed and IMB-301 was added
the supernatants were collected and filtered through a 0.45 µm filter
and then used to infect the SupT1 cells (1 × 105) in 96-well plates
SupT1 cells were lysed and firefly luciferase activities were determined using a firefly Luciferase Assay System (Promega)
The cytotoxicity of IMB-301 was measured using the CCK8 Assay Kit (Beyotime)
The kit provides an assay that distinguishes metabolically active cells from injured cells and dead cells
SupT1 or H9 cells were treated with IMB-301 at various concentrations
DMSO treated cells were used as the control
the samples were subjected to Live/Dead Cell Vitality Assay Kit following the manufacturer manual
the cells were co-transfected with 1 µg Vif and 3 µg hA3G-HA
the cells were treated with DMSO or 10 µM IMB-301 for 24 hr
the cells were collected and lysated in NP-40-containing buffer for 30 min on ice
Cell lysates were centrifugated at 10,000x g for 10 min at 4 °C and supernatant was transferred to a fresh 1.5 ml tube on ice
The cell lysates were incubated with anti-HA rabbit antibody for 3 hr at 4 °C
Thirty microliters of proteinA-Agarose (Santa Cruz) was added and incubated overnight at 4 °C
The beads were washed 4 times with lysis buffer (cold) and at last boiled in 40 µl sample buffer for 5 to 10 min
The samples were analyzed by Western blotting
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USA) for providing the candidate compounds
This work was supported by the National Key Research and Development program of China (2016YFD0500307 CS)
The National Natural Science Foundation of China (81271844 ZJM
National Mega-project for Innovative Drugs (2012ZX09102101-018 CS)
CAMS Innovation Fund for Medical Sciences (CAMS-I2M-1-012 ZJM)
National Mega-Project for Significant new drug discovery (2018ZX09711003-002-002
CAMS Innovation Fund for Medical Sciences (2016-I2M-2-002 LX)
Beijing Key Laboratory of Emerging Infectious Diseases (to WJ) and Xiehe Scholar to CS
Chinese Academy of Medical Sciences &amp; Peking Union Medical College
performed the major part of the experimental study
All authors analyzed the results and approved the final version of the manuscript
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
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DOI: https://doi.org/10.1038/s41598-018-26318-3
Molecular and Cellular Biochemistry (2019)
À vif! is a <a href="/articles/tag/com%C3%A9die-dramatique">comedy-drama</a> directed by John Wells, released in 2015. The film stars <a href="/articles/tag/bradley-cooper">Bradley Cooper</a>, <a href="/articles/tag/sienna-miller">Sienna Miller</a> and  <a href="/articles/tag/daniel-br%C3%BChl">Daniel Brühl</a> in an intense exploration of the world of haute gastronomy
known for his roles in Happiness Therapy and American Sniper
With Sienna Miller and Daniel Brühl at his side
plunges viewers into the ruthless world of Michelin-starred cuisine
was crowned with two Michelin stars before his arrogance and excess led to his downfall
he spent several years trying to regain his sobriety
he surrounded himself with a team of talented young chefs to reopen a restaurant in London
with the ultimate goal of obtaining a third Michelin star
the ghosts of his past and the immense pressure of his ambition make his quest for redemption harder than expected
He now has just one more chance to prove that he can become a true gastronomic legend
will appeal to fans of culinary dramas and those fascinated by the demanding world of haute gastronomy
The film stands out for its realistic and passionate portrayal of Michelin-starred cuisine
with cooking scenes that are as intense as they are captivating
Charismatic performances by Bradley Cooper and Sienna Miller add emotional depth to this story of redemption and perseverance
offers a fascinating and moving immersion in the world of great chefs
is an intense and emotional dive into the world of gastronomy
driven by Bradley Cooper's magnetic performance
this film promises to captivate viewers with its blend of drama
BetweenAdam Jones' personal and professional challenges
this quest for culinary perfection and redemption offers a rich and inspiring cinematic experience
<a href="/en/what-to-do-in-paris/cinema-series/guides/311035-prime-video-new-amazon-originals-and-exclusive-films-and-series-in-june-2024" data-id="311035"> Prime Video: new Amazon Originals and Exclusive films and series in June 2024</a>In June, explore Amazon Originals and Exclusives on Prime Video. <a href="/en/what-to-do-in-paris/cinema-series/guides/311035-prime-video-new-amazon-originals-and-exclusive-films-and-series-in-june-2024">[Read more]</a>
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we are excited to announce the availability of AWS Outposts private connectivity
the service link endpoints in the region for each AWS Outposts deployment were in the public AWS realm of a customer’s chosen region and could be connected to by either the public internet or an AWS Direct Connect (DX) public virtual interface (VIF)
using AWS Outposts public service link connectivity meant that customers needed to enable a connection from the AWS Outposts rack out through their edge routers and firewalls to the service link&nbsp;public endpoint IPs
The service link is important as it is a group of encrypted tunnels that are used for carrying management traffic and your intra Amazon VPC traffic between your chosen AWS Region and AWS Outposts
Service link establishment is required before an Outpost can be used
and must be maintained in order for AWS Outposts to continue to operate
For an overview of AWS Outposts network connectivity, including public service link access, see the <a href="https://d1.awsstatic.com/architecture-diagrams/ArchitectureDiagrams/outposts-reference-architecture-ra.pdf?did=wp_card&amp;trk=wp_card">AWS Outposts – Network Reference Architecture</a>
The new AWS Outposts service link private connectivity feature uses AWS Direct Connect
changing the endpoint for the service link from a public AWS endpoint to a set of private elastic network interfaces (ENIs) within an Amazon VPC deployed in your environment
This means that the service link connects from your AWS Outposts privately over a DX private VIF
to the private AWS Outposts service link endpoints in the Region
Using a private VIF with your Virtual Private Gateway (VGW)
and being attached to an Amazon VPC that you manage
allows you to connect privately to the Outposts service link endpoints without having to traverse the public internet
It additionally gives you the ability to use DX features to troubleshoot connectivity
such as Direct Connect Amazon CloudWatch metrics
Using the AWS Outposts private connectivity option for the service link also removes the need for using large public allow-lists on your on-premises firewall edge
as the service link endpoints are in a VPC that you control
using private addresses that you have allocated to your Amazon VPC
Figure 1. AWS Outposts private connectivity and the Virtual Private Gateway. Note for simplicity only one DX connection is shown. We recommend reviewing the <a href="https://aws.amazon.com/directconnect/resiliency-recommendation/">DX resiliency recommendations</a> to select a DX architecture that meets your availably requirements
is an AWS Outposts (A) installed at your premises with a service link connection (B) to the Outposts service endpoints (C) in the AWS region (D) on the far left
we’re using the VGW (E) attached to your Amazon VPC (F) that terminates a DX private VIF (G) and serves as a private endpoint for the service link connections in the Region for your AWS Outposts
we do not support VPN or AWS Transit Gateway for AWS Outposts private connectivity
you may however want to use an Amazon Direct Connect gateway if needed for cross-region DX access
The DX private VIF&nbsp;is a private connection to your edge router in your chosen DX location and uses BGP to exchange routes
Your private Amazon VPC CIDR range is advertised through this BGP session to your edge router (10.2.0.0/16 in the Figure 1
the /26 IP address range for the Outpost service link (10.5.0.0/26 in the Figure 1
example) is advertised to the region via BGP from your edge router
Note: It is required that your service link endpoint VPC not use the range 10.1.0.0/16 for it’s Amazon VPC CIDR range
and that the Amazon VPC and subnet used be created in the same AWS account
and Availability Zone as your AWS Outposts
you will get the option to select private connectivity for the service link
Once you select the private connectivity option
AWS automatically creates a private connectivity endpoint and assigns private IPs to it from the VPC subnet’s CIDR that you have selected to use for the AWS Outposts private connectivity
all service link traffic between your AWS Outposts rack and the AWS Outposts service endpoints in the Region will use your designated private connectivity
For the service link private endpoint in your VPC, you can configure network ACLs (Access Control Lists) if needed, for the subnet that hosts the&nbsp;private service link endpoints.&nbsp;Traffic to this subnet can be restricted to TCP/UDP source/destination ports 443 as an example, see <a href="https://docs.aws.amazon.com/outposts/latest/userguide/outposts-requirements.html">service link firewall requirements</a> for more information on ACL configuration if needed
Having a dedicated subnet for&nbsp;the&nbsp;private connectivity endpoint offers the advantage of a defining a single ACL to simplifying management and control of traffic related to the Outposts service link
and dedicating a VPC for the service link private endpoints can also help minimize any Outposts service interruptions from other configuration actions in the future
The following tutorial guides you through deploying a new AWS Outposts with private connectivity
must not conflict with 10.1.0.0/16) in the same AWS account and Availability Zone as your Outpost
DX transport between the on-premises Outpost location and the AWS region with a Private VIF connection into the VPC
Advertise the subnet CIDR to your on-premises network
Start by configuring some environment variables
The selection for the private connectivity must be made when creating or provisioning your Outpost
simply select the private connectivity option
Then select the VPC and the Subnet where AWS will automatically create private connectivity endpoints for connecting to the Outpost service
Outposts needs permission to create cross-account network interfaces and attach them to service link endpoint instances
so you have to allow us to create a new role to this
you can see the all the configurations that will be applied your behalf
Finally you will have access to the dashboard where you can view the Outposts summary as well as the connectivity type configured
A service role will be created in the process
Maintaining the Outposts service link connection up
is critical for management of the Outposts
Ensure you have redundant connections to region in case of failure
The private VPCs deployed in your VPC are fully under your control
in order to avoid an unintendedly drop in the service link assign a service control policy that limits deletion of the following resources:
Note: If you decide on leveraging a TGW for the service link
Now when your Outpost connects back to the associated AWS Region
it will use the private service link endpoints in a VPC of your choosing when establishing the AWS Outposts service link connection
Private connectivity provides an additional option when public connectivity is not desired
The public connectivity option for the Outposts service link is still available and supported by AWS Outposts
if limiting your on-premises access to public networks
and using an DX private VIF for AWS Outposts service link private connectivity is something that your organization needs
2024: An earlier version of this post misstated that AWS Outposts operation modes can be changed between public and private connectivity by opening a support ticket
The post has been updated with the incorrect section removed
2025: The section “Best Practices” was added.&nbsp;
AWS Direct Connect now supports connections to AWS Transit Gateway <a href="https://aws.amazon.com/about-aws/whats-new/2022/08/aws-direct-connect-expands-transit-gateway-support-connection-speeds/">at speeds of 500 megabits per second (Mbps) and lower</a>
The architecture described in this post is no longer needed
<a href="https://aws.amazon.com/transit-gateway/" target="_blank" rel="noopener noreferrer">AWS Transit Gateway</a> provides you with the ability to connect multiple <a href="https://docs.aws.amazon.com/vpc/latest/tgw/tgw-vpc-attachments.html" target="_blank" rel="noopener noreferrer">VPCs</a>, <a href="https://docs.aws.amazon.com/vpc/latest/tgw/tgw-vpn-attachments.html" target="_blank" rel="noopener noreferrer">VPNs</a> and scale up to 5,000 attachments
It simplifies management and reduces operational costs of networks within your AWS environments and connectivity from on-premises networks
also known as a sub-1 Gbps hosted AWS Direct Connect connection
I explain how to integrate a sub-1 Gbps hosted Direct Connect connection with AWS Transit Gateway without a transit virtual interface by using the following methods:
Readers of this blog post should be familiar with Border Gateway Protocol (BGP) and the following AWS services:
This method is similar to attaching a VPN to AWS Transit Gateway
you establish a VPN to AWS Transit Gateway over AWS Direct Connect
The following diagram depicts the scenario and the solution
Figure 1: Connecting to transit gateway over a public VIF
Because this is a hosted connection, you don’t have to create a connection in your AWS Direct Connect console. Your <a href="https://aws.amazon.com/directconnect/partners/" target="_blank" rel="noopener noreferrer">AWS Direct Connect Partner</a> creates and delegates the connection to your account
This architecture works well for point-to-point connections between AWS and the customer’s on-premises network
it proves suboptimal for scenarios where the customer’s network consists of multiple sites connected over an MPLS network in a fully meshed manner
MPLS L3 VPN provides the flexibility of connecting multiple sites privately
you must use a Provider Edge (PE) router managed by the MPLS service provider
Due to the scale and multi-tenant nature of these PE routers
VPN tunnels are generally not configured on PE routers as that increases complexity and poses operational risks to this layer
You can use dynamic or static routing for integrating MPLS L3 VPNs to AWS Transit Gateway
The following diagram depicts the architecture used to integrate AWS Transit Gateway to a sub-1 Gbps hosted Direct Connect connection using a dynamic routing protocol
Figure 2: Connecting to AWS Transit Gateway over a private virtual interface via L3 MPLS using BGP
The following steps provide end-to-end connectivity between on-premises networks and VPCs behind an AWS Transit Gateway
This architecture also manages the failover dynamically using BGP
Multiple sets of BGP peering enable prefix exchanges between VPCs and on-premises networks
The following list outlines the BGP peerings:
you don’t have to create a connection in your AWS Direct Connect console for this hosted connection
&nbsp;The AWS Direct Connect Partner creates and delegates the connection to your account
Figure 4: Connecting floating virtual private gateway to AWS Transit Gateway using BGP via Edge Transit VPC
you will have end-to-end connectivity between VPCs and on-premises networks
This option uses static routing between the EC2 instance running a router AMI in the edge transit VPC and the transit gateway
It does not use VPN tunnels between EC2 instances with router AMI and AWS Transit Gateway
allowing you to optimize cost by not incurring cost for the VPN connection
This option still uses VPN tunnels between the floating virtual private gateway and EC2 instance running a router AMI
The following diagram depicts this scenario and the steps to deploy it
Figure 5: Connecting AWS Transit Gateway over a private VIF via L3 MPLS using static routes in transit gateway
follow steps 1 through 6 under Option1: Using dynamic routing protocol between transit gateway and MPLS L3 VPN and then follow these additional steps
Figure 6: Connecting floating virtual private gateway to AWS Transit Gateway via Edge Transit VPC using static routes
or use automation to detect a failover and then switch the paths by modifying the route tables programmatically
I described various ways of integrating AWS Transit Gateway with your on-premises networks over sub-1 Gbps hosted AWS Direct Connect using point-to-point and MPLS L3 VPN
I hope this blog post helps you use AWS Transit Gateway with sub-1 Gbps hosted AWS Direct Connect connection and simplify the connectivity with your AWS environment
The coexistence of two parties with opposing interests &ndash; in this case
lentiviruses for replication and the host that attempts to evade viral infection &ndash; has led to the ongoing battle between the host and virus playing out on a molecular scale over evolutionary time
A3G (also known by its longer acronym APOBEC3G) is a protein that prevents HIV from hijacking host cellular machinery to replicate its genetic material
the A3G protein gets packaged into HIV virions to block viral replication
the viral protein Vif destroys A3G to prevent it from getting packaged into virions in the first place
both A3G and Vif have evolved to outsmart each other
resulting in an ongoing molecular arms race
Scientists have known about A3G and Vif&rsquo;s molecular arms race for decades, but the structural basis of this interaction remained unknown. In a new study published in <a href="https://www.nature.com/articles/s41586-023-05779-1" target="_blank">Nature</a>
a team of scientists from the Fred Hutchinson Cancer Center and the University of California
San Francisco reported the first cryogenic electron microscopy structure of human A3G bound to HIV-1 Vif
&ldquo;The Vif/A3G story has been at the forefront of the conversation surrounding HIV evolution for over 20 years at this point,&rdquo; said <a href="https://research.fredhutch.org/emerman/en.html" target="_blank">Dr. Michael Emerman</a>
a professor in the Human Biology and Basic Sciences Divisions at the Fred Hutch and a co-author on the study
mutations in these proteins have given us a quasi-roadmap for how this virus family spilled over into hominids
and other labs provided crucial insight into the specificities of this protein interface
the structure was something many tried and failed to resolve for the last decade.&rdquo;
The team behind this paper, including Dr. Yen-Li Li, a postdoc in <a href="https://grosslab.ucsf.edu/" target="_blank">Dr. John Gross&rsquo;s</a> lab at UCSF, and Dr. Caleigh Azumaya, the former associate director of the <a href="https://www.fredhutch.org/en/research/shared-resources/core-facilities/electron-microscopy.html" target="_blank">Electron Microscopy Shared Resource</a> at the Fred Hutch
achieved this feat using cryogenic electron microscopy (cryo-EM)
This technique utilizes an electron microscope
with a beam of electrons as the source of light
to image samples that have been cooled to cryogenic temperatures
cryo-EM can render molecular structures at near-atomic resolution
&ldquo;The first thing that jumped out to us was that the &lsquo;arms-race&rsquo; interface between A3G and Vif that had been predicted from positive selection analysis was indeed the site of interaction between A3G and Vif,&rdquo; said Emerman
Positive selection analyses identify specific sites in the protein that have undergone recurrent changes as a result of selective pressures
likely results from its antagonizing interaction with Vif
Previous work had identified two such sites in the A3G protein
the identity of these sites is known to determine the adaptation of Vif to a new host species
The cryo-EM structure of the site of interaction between A3G and Vif confirmed the prior hypothesis that the region of A3G under positive selection is the site of interaction with Vif
is the presence of RNA at the Vif-A3G interface,&rdquo; said &nbsp;Caroline Langley
a PhD candidate in the Emerman lab and a second author on the paper
The cryo-EM structure revealed a single-stranded RNA molecule at the interface of the Vif and A3G proteins
suggesting that RNA acts as a &ldquo;molecular glue&rdquo; that holds the two proteins together
the cryo-EM structure continued to delight the team with the wealth of information it provided
&ldquo;It was also a surprise that Vif was bound to an A3G dimer,&rdquo; said Langley
The ability of A3G to form dimers is critical to its role as a viral restriction factor
it cannot get packaged into virions to carry out subsequent antiviral activities
it appears that Vif has evolved to target A3G when it poses the largest threat to viral replication.&rdquo;
Although cryo-EM provided the structural data
the subsequent analyses of the structure provided much anticipated answers about the evolutionary relationship between Vif and A3G
&ldquo;This analysis revealed to us that the amino acids in the &lsquo;arms race interface&rsquo; are highly variable and species specific
the identities of the amino acids identified as binding RNA in the structure were highly conserved
hinting that RNA interaction is evolutionarily important for Vif antagonism of A3G,&rdquo; said Langley
This article was first published by the Fred Hutch Cancer Center. <a href="https://www.fredhutch.org/en/news/spotlight/2023/03/bs-li-nature.html">Read the original.</a>
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Scientists find that liver protein inhibits of pertussis toxin
offering a potential new treatment for bacterial respiratory disease
Read more about this recent study from the Journal of Biological Chemistry
Scientists discover that triacylglycerol synthesis enzyme drives lipoproteins secretion rather than lipid droplet storage
Researchers analyze protein and RNA data across 13 cancer types to find similarities that could improve cancer staging
Read about this recent article published in Molecular &amp; Cellular Proteomics
Scientists develop a software tool to categorize microbe  species and antibiotic resistance markers to aid clinical and environmental  research
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The human APOBEC3 family of DNA cytosine deaminases serves as a front-line intrinsic immune response to inhibit the replication of diverse retroviruses
APOBEC3F and APOBEC3G are the most potent factors against HIV-1
HIV-1 viral infectivity factor (Vif) targets APOBEC3s for proteasomal degradation
Here we report the crystal structure of the Vif-binding domain in APOBEC3F and a novel assay to assess Vif-APOBEC3 binding
Our results point to an amphipathic surface that is conserved in APOBEC3s as critical for Vif susceptibility in APOBEC3F
Electrostatic interactions likely mediate Vif binding
structure-guided mutagenesis reveals a straight ssDNA-binding groove distinct from the Vif-binding site
and an ‘aromatic switch’ is proposed to explain DNA substrate specificities across the APOBEC3 family
This study opens new lines of inquiry that will further our understanding of APOBEC3-mediated retroviral restriction and provides an accurate template for structure-guided development of inhibitors targeting the APOBEC3-Vif axis
</a>(a) Schematic of the seven A3 intrinsic immune restriction factors (A3A
The three classes of DNA cytosine deaminase domains
The Z2-cytosine deaminase domains are further classified into three subgroups based on sequence similarity
A3F and A3G are the two most potent A3 proteins and exhibit disparate Vif-binding sites (CD1 for A3G and CD2 for A3F)
(b) SDS–PAGE of the purified A3Fc-CD2 after final size exclusion chromatography
(c) BLI kinetic analysis of A3Fc-CD2 binding to ssDNA
Biotin-labelled ssDNA was coupled to streptavidin-coated biosensors and monitored for binding to purified A3Fc-CD2 at 0
The data was analysed based on a 1:1 binding model using the BLItz Pro software
with the fitted curves shown as grey lines
The ssDNA sequence used in the assay is shown below the sensorgram
with the A3Fc-CD2 deamination site and target DNA cytosine underlined and double underlined
Histogram showing the percent mutation on specific rpoB nucleotide sequences for A3Fc-CD2 and A3G-CD2
Results are expressed as the percentage of total mutations from six independent experiments with at least 20 RifR colonies sequenced for both A3Fc-CD2 and A3G-CD2
Inhibition of HIV-1 Vif binding to A3F or A3G would allow the reactivation of effective host innate immune responses to HIV-1
inhibitors targeting the host side of the A3F–Vif or A3G–Vif interface will be less sensitive to viral mutations and will reduce the probability of HIV-1 variants developing resistance to drug treatments
Here we report the crystal structure of the Vif-binding domain in A3F-CD2
Given that A3F and A3G are the most potent A3 restriction factors
this is a key structure needed for the development of new classes of inhibitors to enhance intrinsic immunity against HIV
We performed site-directed mutagenesis to map out the ssDNA-binding site
which revealed a straight groove for DNA binding
as well as a novel aromatic switch that conferrs nucleotide preferences
we developed the first biophysical HIV-1 Vif-APOBEC3 binding assay
and this allowed us to identify a negatively charged Vif-binding surface distinct from the ssDNA-binding groove
In A3Fc-CD2, the catalytic zinc atom is sequestered by H249, C280 and C283 within the canonical (C/H)-(A/V)-E-(X23–28)-P-C-X2-C cytidine deaminase motif (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/ncomms3593#Fig2">Fig. 2a</a>)
The active site residues superimpose well in all A3 structures
a water molecule is coordinated to the catalytic zinc to complete a tetrahedral geometry
This water molecule is activated to become a nucleophile for deamination of the target deoxycytidine nucleotide
the catalytic water molecule is not observed due to the moderate resolution of the electron density map
loop 3 is shorter and no such protein–protein interactions were detected
Both A3G-CD2 molecular surfaces are shown in the same orientation as panel (a)
(d) Nucleic acid–protein interaction ELISA assay
Alanine scanning mutagenesis of selected A3Fc-CD2 ssDNA-binding site residues
Results are expressed as the mean relative absorbance (+s.d
coli expressed WT A3G-CD2 and A3G-CD2 ‘YYFW’
Histogram showing the percent total mutation on specific rpoB nucleotide sequences
Results are expressed as the percentage of total mutations from six independent experiments with at least 20 colonies sequenced
mutations in the ssDNA-binding site did not affect the overall structure of A3Fc-CD2
thus we predict that differences here may determine target DNA specificity
with a ‘YYFW’ motif (termed the nucleotide specificity box)
the ‘YYFW’ motif is replaced by ‘YYFQ’ and ‘YDDQ’ motifs
We propose that this ‘aromatic switch’ in the nucleotide specificity box determines substrate specificities in A3s
the rpoB hotspots in A3G-CD2 ‘YYFW’ are in excellent agreement with those preferred by A3F
Our Vif-binding assay enabled us to map the HIV-1 Vif-binding interface at single-residue resolution
and provides a foundation for the development of new high-throughput assays
a complementary binding surface on A3F-CD2 would be negatively charged and hydrophobic
</a>(a) BLI kinetic analysis of A3Fc-CD2 binding to refolded full-length HIV-1 Vif
Biotin-labelled HIV-1 Vif was coupled to streptavidin-coated biosensors and monitored for binding to purified A3Fc-CD2 at 0
The data were analysed based on a 1:1 binding model
as only one A3F deamination motif is found on the ssDNA
The calculated fitted curves are shown as grey lines
A number of aromatic and hydrophobic residues
Y269 and F290) are buried at the A3Fc-CD2 core
Residues L263 and S264 (shown in orange) were identified in this study to be not important for Vif binding
The acquired immunodeficiency syndrome that results from HIV-1 infection remains a global health threat
Interactions between host restriction factors and viral antagonists
represent intriguing targets for the development of drugs to restrict viral replication and dissemination
we present the crystal structure of a chimeric A3F C-terminal domain containing the Vif-interaction interface
Our combinatorial approach encompassing structural
biochemical and biophysical studies provides insights into the molecular determinants of ssDNA binding
While our apo A3Fc-CD2 structure clearly shows a straight ssDNA-binding groove
a substrate-induced conformational change may bring these residues into proximity to bind ssDNA
it should be emphasized that our current structural understanding of A3 proteins is limited to the analysis of a single domain of A3 proteins
It may also be possible that ssDNA binds to residues identified in both the ‘straight’ and ‘kinked’ models of DNA binding in the context of full-length or higher-ordered oligomeric A3 protein structures
Our study clearly demonstrates the importance of four residues in the nucleotide specificity box and a switch in hydrophobicities in determining substrate specificities in A3 proteins
suggesting no effects on overall structural stability
We suggest that the hydrophobic site is involved in maintaining the structural integrity and stability of A3Fc-CD2
it may be possible that the hydrophobic Vif-binding site has a role in both protein stability and Vif binding
Full characterization of the role of the hydrophobic site in Vif binding awaits the structural determination of an A3–Vif complex
</a>A negatively charged surface conserved with other Z2-cytosine deaminase domains is proposed to be important for Vif-binding
Electrostatic potential mapped onto the molecular surface of A3C (PDB: 3VOW)
The proposed footprints of the A3 negative and hydrophobic patch involved in Vif binding are shown by the solid and dashed lines
A previously characterized ‘DPD’ motif involved in A3G Vif binding is displayed for the A3G-CD1 homology model
Red and blue coloured regions denote negative and positive charges
Note: the ssDNA-binding site is at the top of the depicted A3 molecules and has no overlap with the hydrophobic or negatively charged Vif-binding site
Our crystal structure presented in this manuscript has now identified well-defined sites on A3Fc-CD2 involved in ssDNA and Vif binding
We have also identified novel structural determinants that explain the differences in substrate specificities between A3 family members
our studies and findings will be invaluable to the A3 community by providing the relevant structural scaffold for the development of effective HIV-1 inhibitors aimed at selectively disrupting the A3F-Vif interface
The structures of A3A and A3F11X-CD2 align well with A3Fc-CD2
no conformational changes in the chimeric region of A3Fc-CD2 exist
A3Fc-CD2 and mutants were expressed in Rosetta-2 (DE3) E
Cell cultures were grown to OD600=0.8 and induced with a final concentration of 0.5 mM IPTG for 18 h at 25 °C
Cells were resuspended in Ni-binding buffer (50 mM Tris-HCl pH 8.0
300 mM NaCl and 20 mM imidazole) with EDTA-free protease inhibitor cocktail and lysed at 30 kpsi using a hydraulic cell disruption system (Constant Systems TS benchtop)
The lysate was centrifuged to remove cellular debris prior to loading onto Ni-NTA resin (Thermo Pierce)
A3Fc-CD2 was washed with Ni-NTA binding buffer with 50 mM imidazole
A3Fc-CD2 was eluted by a two-step gradient of Ni-binding buffer with 125 mM imidazole and Ni-binding buffer with 500 mM imidazole
Fractions that contained A3Fc-CD2 were concentrated and purified on a Superdex-200 10/300 GL column equilibrated in Buffer A (10 mM Tris-HCl pH 7.5
A3Fc-CD2 was further purified by anion exchange chromatography
Here A3Fc-CD2 was pooled and loaded onto a MonoQ HR 5/5 column equilibrated in Buffer B (10 mM Tris-HCl pH 7.5
A3Fc-CD2 was eluted using a linear gradient of 0–100% Buffer B with 1 M NaCl
Purified A3Fc-CD2 was quantified by A280 and concentrated to 20 mg ml−1 for crystallization
All molecular ribbon diagrams and vacuum electrostatic calculations were generated using the programme MacPyMOL
This ssDNA has one A3F deamination site (5′-CTCA-3′)
Biotinylated ssDNA was diluted to 50 μM in kinetics buffer (PBS
immobilized on a streptavidin biosensor for 120 s
20 μM of biotinylated ssDNA was immobilized on the probe
A3Fc-CD2 was diluted into kinetics buffer (0
2 and 4 μM) and association to the biotinylated ssDNA was measured over 300 s
the biosensor was immersed in kinetics buffer for 300 s to measure dissociation
The Kd was calculated using the BLItz Pro v.1.1.0.28 software
Single-stranded 42-mer DNA (<a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/ncomms3593#Fig1">Fig. 1c</a>) was synthesized (Integrated DNA Technologies)
Biotinylated ssDNA (1.5 pmol) was immobilized onto a streptavidin-coated ELISA plate (Quidel) and washed three times with PBS with 0.01% (v/v) Tween-20 (PBS-T)
Wells were blocked with 3% (w/v) BSA in PBS-T overnight at 4 °C
75 and 100 μg of A3Fc-CD2 or A3Fc-CD2 mutants were incubated at 22 °C for 1.5 h and subsequently washed three times with PBS-T
A mouse anti-His primary mAb (Roche) diluted 1:3,000 in PBS-T was incubated for 1 h at 22 °C
The plate was washed three times prior to incubation with a goat anti-mouse HRP-conjugated secondary mAb (Pierce) diluted 1:3,000
The plates were developed using TMB-One substrate solution (Kem-En-Tec Diagnostics) for 5 min
Colour development was stopped with 2 N sulphuric acid and measured at 495 nm
the biotinylated sense and antisense DNA strands (3′-AGG GAG TCT GGG AAA ATC AGT CAC ACC TTT TAG AGA TCG TCA-5′) were heated at 95 °C for 15 min and allowed to cool to 22 °C in annealing buffer (10 mM Tris-HCl (pH 7.5)
The double-stranded DNA probe was then immobilized onto the streptavidin-coated ELISA plate and performed as described above
All experiments were performed in triplicate
A3Fc-CD2 or A3Fc-CD2 ssDNA-binding mutants cloned into pET46-Ek/LIC were transformed into BL21-(DE3) E
Single colonies were used to inoculate a 10 ml culture of LB supplemented with 100 μg ml−1 ampicillin
the cultures were induced with 1 mM final concentration of IPTG and grown overnight at 37 °C
Cell cultures were normalized (7 ml of cells at OD600=0.5) and plated onto LB-agar plates containing 100 μg ml−1 rifampicin to select for RifR clones
Single colonies were picked from plates and colony PCR was performed to amplify the bacterial RNA polymerase gene rpoB
The rpoB PCR products were DNA sequenced with an rpoB sequencing primer (5′-GGC-GAA-ATG-GCG-GAA-AAC-3′)
A3F or A3G DNA-binding motifs in the rpoB gene were analysed for C–to-T mutations
CD wavelength scans and thermal titrations were performed on all A3Fc-CD2 proteins at a concentration of 1.0 mg ml−1 in PBS
0.05% (w/v) CHAPS and 1 M guanidine-HCl on a Jasco J-810 spectropolarimeter
CD wavelength scans collected between 190–250 nm using a 1-mm quartz cuvette (Helma) were averaged over five scans
Thermal denaturation of A3Fc-CD2 and mutants were performed by increasing the temperature from 20–95 °C and monitoring the loss in CD signal at 222 nm
HIV-1 Vif was expressed in BL21-(DE3) E. coli cells, purified and refolded from inclusion bodies, as previously described<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 32" title="Gallerano, D. et al. Biophysical characterization of recombinant HIV-1 subtype C virus infectivity factor. Amino Acids 40, 981–989 (2011)." href="/articles/ncomms3593#ref-CR32" id="ref-link-section-d454875379e2781">32</a>
Vif inclusion bodies were resolubilized in 6 M guanidine–HCl and 10 mM Tris–HCl (pH 7.4) overnight at 22 °C
and subsequently clarified by centrifugation prior to Ni-NTA purification
The Ni-NTA column was washed with 8 M urea
100 mM NaH2PO4 and 10 mM Tris-HCl (pH 6.9)
and Vif was eluted in the same buffer at pH 4.5
Vif was subsequently purified on a prep grade Superdex-75 10/300 column equilibrated in 8 M urea
10 mM β-mercaptoethanol and 10 mM Tris-HCl pH 4.5
The peak corresponding to monomeric Vif was collected and diluted to 0.1 mg ml−1 and dialyzed against 100 mM NaH2PO4
20% (v/v) glycerol (pH 6.0) with decreasing concentration of urea (6
The final refolded HIV-1 Vif was dialyzed against 10 mM NaH2PO4 pH 6.0
and biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation kit (Thermo Pierce) according to the manufacturer’s protocol
Biotinylated Vif was diluted to 40 μg ml−1 in 10 mM NaH2PO4
1 mg ml−1 BSA and 0.002% (v/v) Tween-20 and immobilized onto a BLI streptavidin probe for 120 s
A3Fc-CD2 mutants were diluted into 10 mM Tris-HCl (pH 7.5)
1 mg ml−1 BSA and 0.002% (v/v) Tween-20 and allowed to associate over 80 s
Purified A3G-CD2 was used as a negative control
Accession codes: Atomic coordinates and structure factors for A3Fc-CD2 have been deposited in the Protein Data Bank (PDB) with the accession code <a href="http://www.pdb.org/pdb/search/structidSearch.do?structureId=4J4J">4J4J</a>
An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22
Quantitative profiling of the full APOBEC3 mRNA repertoire in lymphocytes and tissues: implications for HIV-1 restriction
APOBEC3G multimers are recruited to the plasma membrane for packaging into human immunodeficiency virus type 1 virus-like particles in an RNA-dependent process requiring the NC basic linker
HIV-1 Vif alters processive single-stranded DNA scanning of the retroviral restriction factor APOBEC3G
Multiple ways of targeting APOBEC3-virion infectivity factor interactions for anti-HIV-1 drug development
interaction and real-time monitoring of the enzymatic reaction of wild-type APOBEC3G
Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications
Crystal structure of the APOBEC3G catalytic domain reveals potential oligomerization interfaces
Rationalisation of the differences between APOBEC3G structures from crystallography and NMR studies by molecular dynamics simulations
APOBEC2 is a monomer in solution: implications for APOBEC3G models
Dissecting APOBEC3G substrate specificity by nucleoside analog interference
Biophysical characterization of recombinant HIV-1 subtype C virus infectivity factor
Single-strand specificity of APOBEC3G accounts for minus-strand deamination of the HIV genome
Mutational comparison of the single-domained APOBEC3C and double-domained APOBEC3F/G anti-retroviral cytidine deaminases provides insight into their DNA target site specificities
Hydrophobicities of the nucleic acid bases: distribution coefficients from water to cyclohexane
An automated microseed matrix-screening method for protein crystallization
The finer things in X-ray diffraction data collection
Protein structure prediction on the Web: a case study using the Phyre server
A new bioinformatics analysis tools framework at EMBL-EBI
ENDscript: a workflow to display sequence and structure information
<a data-track="click" data-track-action="download citation references" data-track-label="link" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/ncomms3593?format=refman&amp;flavour=references">Download references</a>
This work was supported by an Ontario HIV Treatment Network Operating Grant (ROG-G645)
Canada Research Chair in Structural Virology and a Canadian Institutes of Health Research (CIHR) New Investigator Award (MSH-113554) to J.E.L
A CIHR Postdoctoral Fellowship was awarded to K.K.S.
was supported by a University of Toronto Graduate Scholarship and CIHR Masters Graduate Fellowship
We are indebted to Douglas Instruments (UK) for the generous use of their Oryx 8 crystallization robot for microseeding and Aiping Dong
Professors Cheryl Arrowsmith and Aled Edwards from the Structural Genomics Consortium (University of Toronto) for access to their X-ray diffraction facility
This work is based upon research conducted at the NE-CAT 24-ID-E beamline (Advanced Photon Source
supported by award RR-15301 from the National Center for Research Resources (NIH)
Use of the APS is supported by the US Department of Energy
Department of Laboratory Medicine and Pathobiology
conceived and supervised the project; K.K.S.
crystallization and structure determination; A.S
bacterial RifR DNA cytosine deaminase assays and CD spectroscopy; F.C.A
purification and refolding of HIV-1 Vif for binding studies; K.K.S
Supplementary Table S1 and Supplementary Discussion (PDF 1082 kb)
<a data-test="citation-link" data-track="click" data-track-action="download article citation" data-track-label="link" data-track-external="" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/ncomms3593?format=refman&amp;flavour=citation">Download citation</a>
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The essential HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells expressing cytidine deaminases APOBEC3G (A3G) and A3F by decreasing their cellular level
and preventing their incorporation into virions
the functional role of the inhibition of A3G translation by Vif remained unclear
we show that two stem-loop structures within the 5′-untranslated region of A3G mRNA are crucial for translation inhibition by Vif in cells
and most Vif alleles neutralize A3G translation efficiently
K26R mutation in Vif abolishes degradation of A3G by the proteasome but has no effect at the translational level
indicating these two pathways are independent
proteasomal degradation and translational inhibition
similarly contribute to decrease the cellular level of A3G by Vif and to prevent its incorporation into virions
inhibition of A3G translation is sufficient to partially restore viral infectivity in the absence of proteosomal degradation
These findings demonstrate that HIV-1 has evolved redundant mechanisms to specifically inhibit the potent antiviral activity of A3G
the relative importance of the translational inhibition of A3G by Vif
compared to the well-documented A3G degradation
and its impact on viral infectivity remained to be established
we used several A3G mRNA expression plasmids mutated in their UTRs
with and without inhibitors of A3G degradation by the proteasome
Our data show that two stem-loop structures in the 5′-UTR of A3G mRNA are required for translational inhibition by Vif
The property of Vif to inhibit the translation of A3G is common to a large variety of Vif alleles and was also demonstrated in HIV-1 chronically infected H9 cells
which abolishes degradation of A3G by the proteasome but has no effect on the translational repression of A3G
demonstrating that these two pathways are independent
These two mechanisms contribute to the decrease of the intracellular level of A3G by Vif and to the subsequent A3G incorporation into virions
the inhibition of A3G translation by Vif is sufficient to partially restore viral infectivity in A3G expressing cells in the absence of proteasomal degradation
These findings demonstrate that HIV-1 has evolved several redundant mechanisms to specifically inhibit the potent antiviral activity of A3G proteins
</a>Schematic representation of A3G constructs used in this study.
Wild-type authentic A3G mRNA and mutants deleted from their 5′, 3′ or 5′ and 3′-UTRs are represented. Secondary structures of the 5′- and 3′UTRs of wild-type A3G mRNA are also indicated with high affinity binding sites for Vif depicted in grey. Dotted lines represent the deletions.
</a>Vif inhibits A3G translation in a 5′UTR dependent manner
All samples derive from the same experiment and blots were processed in parallel
</a>Vif inhibits A3G translation in HIV-1 chronically infected H9 cells
</a>Heterologous 5′UTRs do not allow inhibition of A3G translation by Vif
</a>Vif requires SL2 and SL3 to impair A3G translation
</a>Vif K26 residue is required for the translational inhibition of A3G
</a>Effect of the inhibition of A3G translation by Vif on A3G packaging and viral infectivity
these two mechanisms significantly contribute to exclude A3G from viral particles
the amount of Vif protein encapsidated into viral particles was constant under all conditions studied
indicating that a direct competition between Vif and A3G for packaging is unlikely
These results indicate that the inhibition of A3G translation by Vif is sufficient to partially restore HIV-1 infectivity
</a>Translational repression of A3G by different Vif alleles
HEK 293T cells were transfected with plasmids expressing wild-type A3G mRNA in the presence of Vif alleles and proteasome inhibition (ALLN)
and the relative A3G expression was analyzed by western blot and quantify using Image J (1.46r)
Standard deviations are representative of at least three independent experiments
raising the possibility that Vif/A3G interaction might be required for translational regulation
suggesting that specific sequences/domains of Vif are required to down-regulate A3G translation
This translational control is corroborated by the fact that this property is shared by almost all Vif proteins and opens attractive perspectives for the development of new drugs disrupting the translational control of A3G by Vif
considering that the 5′UTR of A3G and A3F mRNAs is highly conserved
it is likely that Vif is also able to inhibit A3F translation
into the double digested pCMV A3G expression vector
All constructs were confirmed by DNA sequencing (GATC Biotech
HEK 293T cells were co-transfected with pA3G-HA and plasmids expressing wild type or mutant Vif
0.05% SDS) supplemented with protease inhibitors (cOmplete EDTA Free cocktail
an aliquot fraction (50 μl) was used for determination of the protein expression level
and the remaining was incubated 2 h at 4 °C with 1 μg of HA antibody (Santa Cruz
protein A Dynabeads (Life Technologies) were added and incubated for 1.5 h at 4 °C
NuPAGE LDS sample buffer (Life Technologies)
supernatant was loaded on NuPAGE gel (Life Technologies) and analyzed by western blot
Polyclonal anti-hA3G (#9968) and monoclonal anti-HIV-1 Vif (#319) antibodies were obtained through the NIH AIDS Research and Reference Reagent Program
Monoclonal anti-β-actin antibody was purchased from SIGMA (#A5316)
An HIV-positive patient serum was used for the identification of HIV-1 p24 protein
The PVDF membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (BIO-RAD)
and the proteins were visualized by enhanced chemiluminescence (ECL) using the ECL Prime Western blotting detection reagent (GE Healthcares)
Bands were quantified using Image J (1.46r) by analyzing pixel density
Student’s T-test was used to determine statistical significance
5.106 cells (treated or not with ALLN/DMSO) were harvested by centrifugation and lysed for 10 min at 4 °C in RIPA 1X supplemented with protease inhibitors
Lysates were cleared by centrifugation for 30 min at 14,000 g and protein concentration was determined using a Bradford assay in order to load the equivalent of 150 μg of total proteins on a NuPAGE® Novex® 4–12% Bis-Tris gels (Life Technologies)
Western blot was then performed as above using antibodies directed against A3G (NIH#9968)
GAPDH (ABD Serotec-Bio-Rad); p24 (HIV-positive patient serum) and Ubiquitin (Santa Cruz
Appropriate HRP-conjugated secondary antibodies were used and revealed by chemiluminescence
total RNA was isolated from 293T cells using TRI Reagent (SIGMA)
total RNA was isolated by phenol/chloroform extraction followed by ethanol precipitation
Total RNA (1 μg) was then reverse-transcribed using the iScriptTM Reverse Transcription Supermix (BIO-RAD) as recommended by the manufacturer
Subsequent qPCR analysis was performed using the KAPA SYBR® FAST qPCR Master Mix (KAPA BIOSYSTEMS) and was monitored on a CFX Real Time System (BIO-RAD)
Gene-specific primers were: A3G forward primer
and reverse primer 5′-TTCCAAAAGGGAATCACGTC-3′; β-actin forward primer
and reverse primer 5′-AGCACTGTGTTGGCGTACAG-3′
The A3G mRNA levels were normalized to those of β-actin mRNA and relative quantification was determined using the standard curve based method
Translational regulation of APOBEC3G mRNA by Vif requires its 5′UTR and contributes to restoring HIV-1 infectivity
The restriction factors of human immunodeficiency virus
Tumultuous relationship between the human immunodeficiency virus type 1 viral infectivity factor (Vif) and the human APOBEC-3G and APOBEC-3F restriction factors
APOBEC proteins and intrinsic resistance to HIV-1 infection
Remarkable lethal G-to-A mutations in vif-proficient HIV-1 provirus by individual APOBEC3 proteins in humanized mice
APOBEC3G is incorporated into virus-like particles by a direct interaction with HIV-1 Gag nucleocapsid protein
HIV-1 and MLV Gag proteins are sufficient to recruit APOBEC3G into virus-like particles
The role of innate APOBEC3G and adaptive AID immune responses in HLA-HIV/SIV immunized SHIV infected macaques
Suppression of HIV-1 infection by APOBEC3 proteins in primary human CD4(+) T cells is associated with inhibition of processive reverse transcription as well as excessive cytidine deamination
APOBEC3F/G and Vif: action and counteraction
Host restriction factors in retroviral infection: promises in virus-host interaction
Identification of the HIV-1 Vif and Human APOBEC3G Protein Interface
The Binding Interface between Human APOBEC3F and HIV-1 Vif Elucidated by Genetic and Computational Approaches
Vif hijacks CBF-β to degrade APOBEC3G and promote HIV-1 infection
CBF-β stabilizes HIV Vif to counteract APOBEC3 at the expense of RUNX1 target gene expression
Differential requirements for HIV-1 Vif-mediated APOBEC3G degradation and RUNX1-mediated transcription by core binding factor beta
Identification of HIV-1 Vif regions required for CBF-β interaction and APOBEC3 suppression
The human immunodeficiency virus type 1 Vif protein reduces intracellular expression and inhibits packaging of APOBEC3G (CEM15)
The mechanism of eukaryotic translation initiation and principles of its regulation
Regulation of mRNA translation by 5′- and 3′-UTR-binding factors
HIV-1 replication and the cellular eukaryotic translation apparatus
Stoichiometry of the antiviral protein APOBEC3G in HIV-1 virions
Highly purified human immunodeficiency virus type 1 reveals a virtual absence of Vif in virions
Multiple lysines combined in HIV-1 Vif determines the responsiveness to CBF-beta
Identification of an APOBEC3G binding site in human immunodeficiency virus type 1 Vif and inhibitors of Vif-APOBEC3G binding
Evidence that ecotropic murine leukemia virus contamination in TZM-bl cells does not affect the outcome of neutralizing antibody assays with human immunodeficiency virus type 1
Viral RNA is required for the association of APOBEC3G with human immunodeficiency virus type 1 nucleoprotein complexes
Effects of lysine to arginine mutations in HIV-1 Vif on its expression and viral infectivity
Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone
Cytoskeleton association and virion incorporation of the human immunodeficiency virus type 1 Vif protein
Different effects of the TAR structure on HIV-1 and HIV-2 genomic RNA translation
HIV-1 gp41-specific monoclonal mucosal IgAs derived from highly exposed but IgG-seronegative individuals block HIV-1 epithelial transcytosis and neutralize CD4(+) cell infection: an IgA gene and functional analysis
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Redmond Smyth for critical reading of the manuscript
France) who kindly provided us vectors expressing the different Vif alleles and vectors expressing heterologous 5′UTR
The following reagents were obtained through the AIDS Research and Reference Reagent Program
NIH: A3G polyclonal antibody (#9968) from Dr
Warner Greene and Vif monoclonal antibody (#319) from Dr
This work was supported by a grant from the French National Agency for Research on AIDS and Viral Hepatitis (ANRS) and SIDACTION to J.C.P.
and by post-doctoral (J.B.) and doctoral (S.G
also received funding from the Ecuadorian government through the National Secretary of Higher Education
Technology and Innovation (Secretaría Nacional de Educación Superior
Funding for open access charge: French National Center for Research (CNRS)
The Barcelona Institute for Science and Technology
Roland Marquet &amp; Jean-Christophe Paillart
Laboratoire d’ImmunoRhumatologie Moléculaire
Fédération de Médecine Translationnelle de Strasbourg (FMTS)
wrote the paper with contributions from R.M
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using an affinity tag/purification mass spectrometry approach
that Vif additionally recruits the transcription cofactor CBF-β to this ubiquitin ligase complex
which normally functions in concert with RUNX DNA binding proteins
allows the reconstitution of a recombinant six-protein assembly that elicits specific polyubiquitination activity with APOBEC3G
Using RNA knockdown and genetic complementation studies
we also demonstrate that CBF-β is required for Vif-mediated degradation of APOBEC3G and therefore for preserving HIV-1 infectivity
simian immunodeficiency virus (SIV) Vif also binds to and requires CBF-β to degrade rhesus macaque APOBEC3G
Methods of disrupting the CBF-β–Vif interaction might enable HIV-1 restriction and provide a supplement to current antiviral therapies that primarily target viral proteins
HIV-1 accessory proteins—ensuring viral survival in a hostile environment
Interactions of host APOBEC3 restriction factors with HIV-1 in vivo: implications for therapeutics
Getting into position: the catalytic mechanisms of protein ubiquitylation
Recognition of the polyubiquitin proteolytic signal
Purification and characterization of HIV-human protein complexes
Global landscape of HIV–human protein complexes
Nature doi:10.1038/nature10719 (this issue)
VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases
E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification
Interplay of transcription factors in T-cell differentiation and function: the role of Runx
Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin-ligase complex SCF-Cdc34
Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages
APOBEC3 proteins mediate the clearance of foreign DNA from human cells
Sequential E2s drive polyubiquitin chain assembly on APC targets
Priming and extending: a UbcH5/Cdc34 E2 handoff mechanism for polyubiquitination on a SCF substrate
Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation
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Gross and Harris laboratories for comments
Schulman and the AIDS Research and Reference Reagent Program for reagents
This research was funded by grants from QB3 at University of California
and the National Institutes of Health (P50 GM082250
P01 AI090935 and P50 GM081879 to N.J.K.; U54 RR022220 to A.S.; R01 AI064046 and P01 GM091743 to R.S.H.; P50 GM082250 to J.D.G
and C.S.C.; P41RR001614 and P50GM081879 to A.B.)
is a Searle Scholar and a Keck Young Investigator
Hultquist: These authors contributed equally to this work
 California Institute for Quantitative Biosciences
Supplementary References and Supplementary Figures 1-8 with legends
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The transcription cofactor CBF-β (core binding factor β) regulates the DNA binding activity of RUNX family proteins
Two independent studies now show that CBF-β also regulates the ability of HIV-1 to evade host restriction mediated by the cDNA deaminase APOBEC3G
a host factor that blocks viral replication
They show that it associates with the HIV protein Vif
and is essential for the assembly of the Vif-Cul5 E3 ubiquitin ligase complex
which mediates the ubiquitination and destruction of APOBEC3
Both groups suggest that disrupting the Vif–CBF-β interaction could provide a new therapeutic target against HIV-1 infection
<a href="https://www.edsurge.com/organizations/vif-international-education" target="_blank" rel="nofollow noopener">VIF International Education</a>, a K-12 professional development company, has acquired <a href="https://www.participate.com/" target="_blank" rel="nofollow noopener">Participate Learning</a>
a teacher resource aggregator and search engine
Financial terms of the deal were not disclosed
Short for “Visiting International Faculty,” VIF was founded in 1987 to help universities recruit international faculty
and later turned its attention to K-12 educators and bringing international “visiting” teachers to the U.S
The organization later rebranded as VIF International Education in the 1990s
focused on providing more general professional development around developing educators’ global and cultural awareness
VIF International CEO David Young tells EdSurge that he and his team have been interested in turning the organization “into a full-blown edtech company,” where VIF provides educators with the tools they need to do their job
“We needed to train our teachers and provide them with resources to be successful,” VIF International CEO David Young tells EdSurge
adding that VIF currently has 20,000 teachers (1,000 of whom are “visiting international faculty from about 30 countries around the world”) on its platform
Young explains that Participate Learning’s online platform will power VIF’s programming starting in the 2016-20­17 school year
with Participate’s team developing a “better user experience” to sustain “more stability
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