Vif Variants Research Articles

Introduction of Mutant APOBEC3G Into CD4+ T-Cells to Resist HIV-1

Introduction: Human immunodeficiency virus 1 (HIV-1) is a potentially lethal retrovirus with high genetic diversity and replication ability. HIV-1 mainly targets human CD4+ T-cells, causing a rapid decline in CD4+ T-cell count after infection. Apolipoprotein B mRNA Editing Enzyme Catalytic Subunit 3G (APOBEC3G or A3G) is a human anti-viral enzyme that blocks early stages of HIV-1 replication by deaminating the cytosine residues to uracil in the viral minus-strand DNA during reverse transcription. Nevertheless, HIV-1 overcomes A3G anti-viral activity by employing Vif accessory protein to degrade A3G. Due to a proline/aspartate point mutation in residue 129 (P129D), an artificial A3G mutant, also known as A3G-P129D, was found to be resistant against diverse Vif variants, including HIV-1 Vif, thereby shedding light on a potential A3G-mediated gene therapy for HIV-1. Methods: We propose that, using CRISPR-Cas9 tools, the P129D mutation will be introduced into A3G expressed by human hematopoietic stem cells. Engineered stem cells will be transplanted into Non-Obese Diabetic Severe Combined Immunodeficient Gamma (NSG) mice, which will later be infected with HIV-1. Following infection, flow cytometry will be used to compare the CD4+ T-cell count in mice that express A3G-P129D versus control mice transplanted with unmodified human CD4+ T-cells. Results: We expect a notably higher CD4+ T-cell count in mice carrying the P129D mutation than in the control group, which would be attributed to the efficient inhibition of HIV-1 replication by the mutant A3G. Discussion: Transforming human A3G to A3G-P129D will prevent Vif-mediated degradation of A3G, allowing A3G to efficiently target HIV-1. Thus, this treatment would ultimately block HIV-1 replication and prevent CD4+ T-cell depletion. Conclusion: Overall, our novel gene therapy approach could open the door to a potential one-time treatment for HIV-1 infected patients.

Critical role of PP2A-B56 family protein degradation in HIV-1 Vif mediated G2 cell cycle arrest

HIV-1 Vif forms an E3 ubiquitin ligase complex with host proteins to counteract host restrictive APOBEC3, and is also known to accumulate infected cells at the G2 phase to promote viral replication. However, the underlying mechanism of how Vif induces G2 arrest is not fully understood, and more specifically, direct target molecules of G2 arrest have not been identified. Here we show that degradation of B56 family proteins (PP2A-B56), one of the regulatory subunits of protein phosphatase 2A, is critical for the Vif-induced G2 arrest. NL4-3 Vif caused degradation of PP2A-B56, and complementation of PP2A-B56 overcome the Vif-induced arrest. Supportively, knockdown of PPP2R5D, one of PP2A-B56, by siRNA itself induced cell cycle arrest of non-infected cells. We also identified Vif residues I31 and R or K33 are determinants for inducing G2 arrest, and Vif variants that did not cause G2 arrest did not induce PPP2R5D degradation, although it maintain the ability to induce APOBEC3G degradation, showing strong correlation between Vif-induced arrest and PP2A-B56 degradation. In a sequence database of HIV-1 isolates, Vif strains harboring residues that presumably induce cell cycle arrest are approximately 43%, suggesting Vif-induced G2 arrest contributes to HIV-1 infection in vivo and spread. Our data help understand the mechanism of Vif-mediated arrest, and gain insights into general cell cycle regulation.

Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest.

The seminal description of the cellular restriction factor APOBEC3G and its antagonism by HIV-1 Vif has underpinned two decades of research on the host-virus interaction. We recently reported that HIV-1 Vif is also able to degrade the PPP2R5 family of regulatory subunits of key cellular phosphatase PP2A (PPP2R5A-E; Greenwood et al., 2016; Naamati et al., 2019). We now identify amino acid polymorphisms at positions 31 and 128 of HIV-1 Vif which selectively regulate the degradation of PPP2R5 family proteins. These residues covary across HIV-1 viruses in vivo, favouring depletion of PPP2R5A-E. Through analysis of point mutants and naturally occurring Vif variants, we further show that degradation of PPP2R5 family subunits is both necessary and sufficient for Vif-dependent G2/M cell cycle arrest. Antagonism of PP2A by HIV-1 Vif is therefore independent of APOBEC3 family proteins, and regulates cell cycle progression in HIV-infected cells.

Inhibition of Vif-Mediated Degradation of APOBEC3G through Competitive Binding of Core-Binding Factor Beta

Vif counteracts the host restriction factor APOBEC3G (A3G) and other APOBEC3s by preventing the incorporation of A3G into progeny virions. We previously identified Vif mutants with a dominant-negative (D/N) phenotype that interfered with the function of wild-type Vif, inhibited the degradation of A3G, and reduced the infectivity of viral particles by increased packaging of A3G. However, the mechanism of interference remained unclear, in particular since all D/N Vif mutants were unable to bind Cul5 and some mutants additionally failed to bind A3G, ruling out competitive binding to A3G or the E3 ubiquitin ligase complex as the sole mechanism. The goal of the current study was to revisit the mechanism of D/N interference by Vif mutants and analyze the possible involvement of core binding factor beta (CBFβ) in this process. We found a clear correlation of D/N properties of Vif mutants with their ability to engage CBFβ. Only mutants that retained the ability to bind CBFβ exhibited the D/N phenotype. Competition studies revealed that D/N Vif mutants directly interfered with the association of CBFβ and wild-type Vif. Furthermore, overexpression of CBFβ counteracted the interference of D/N Vif mutants with A3G degradation by wild-type Vif. Finally, overexpression of Runx1 mimicked the effect of D/N Vif mutants and inhibited the degradation of A3G by wild-type Vif. Taken together, we identified CBFβ as the key player involved in D/N interference by Vif.IMPORTANCE Of all the accessory proteins encoded by HIV-1 and other primate lentiviruses, Vif has arguably the strongest potential as a target for antiviral therapy. This conclusion is based on the observation that replication of HIV-1 in vivo is critically dependent on Vif. Thus, inhibiting the function of Vif via small-molecule inhibitors or other approaches has significant therapeutic potential. We previously identified dominant-negative (D/N) Vif variants whose expression interferes with the function of virus-encoded wild-type Vif. We now show that D/N interference involves competitive binding of D/N Vif variants to the transcriptional cofactor core binding factor beta (CBFβ), which is expressed in cells in limiting quantities. Overexpression of CBFβ neutralized the D/N phenotype of Vif. In contrast, overexpression of Runx1, a cellular binding partner of CBFβ, phenocopied the D/N Vif phenotype by sequestering endogenous CBFβ. Thus, our results provide proof of principle that D/N Vif variants could have therapeutic potential.

The Structural Interface between HIV-1 Vif and Human APOBEC3H

Human APOBEC3H (A3H) is a cytidine deaminase that inhibits HIV-1 replication. To evade this restriction, the HIV-1 Vif protein binds A3H and mediates its proteasomal degradation. To date, little information on the Vif-A3H interface has been available. To decipher how both proteins interact, we first mapped the Vif-binding site on A3H by functionally testing a large set of A3H mutants in single-cycle infectivity and replication assays. Our data show that the two A3H α-helixes α3 and α4 represent the Vif-binding site of A3H. We next used viral adaptation and a set of Vif mutants to identify novel, reciprocal Vif variants that rescued viral infectivity in the presence of two Vif-resistant A3H mutants. These A3H-Vif interaction points were used to generate the first A3H-Vif structure model, which revealed that the A3H helixes α3 and α4 interact with the Vif β-sheet (β2-β5). This model is in good agreement with previously reported Vif and A3H amino acids important for interaction. Based on the predicted A3H-Vif interface, we tested additional points of contact, which validated our model. Moreover, these experiments showed that the A3H and A3G binding sites on HIV-1 Vif are largely distinct, with both host proteins interacting with Vif β-strand 2. Taken together, this virus-host interface model explains previously reported data and will help to identify novel drug targets to combat HIV-1 infection.IMPORTANCE HIV-1 needs to overcome several intracellular restriction factors in order to replicate efficiently. The human APOBEC3 locus encodes seven proteins, of which A3D, A3F, A3G, and A3H restrict HIV-1. HIV encodes the Vif protein, which binds to the APOBEC3 proteins and leads to their proteasomal degradation. No HIV-1 Vif-APOBEC3 costructure exists to date despite extensive research. We and others previously generated HIV-1 Vif costructure models with A3G and A3F by mapping specific contact points between both proteins. Here, we applied a similar approach to HIV-1 Vif and A3H and successfully generated a Vif-A3H interaction model. Importantly, we find that the HIV-1 Vif-A3H interface is distinct from the Vif-A3G and Vif-A3F interfaces, with a small Vif region being important for recognition of both A3G and A3H. Our Vif-A3H structure model informs on how both proteins interact and could guide toward approaches to block the Vif-A3H interface to target HIV replication.

Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants.

Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function.

Vif Proteins from Diverse Human Immunodeficiency Virus/Simian Immunodeficiency Virus Lineages Have Distinct Binding Sites in A3C.

The APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived A3C, rhA3C and smmA3C, were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm, and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive in the degradation of hA3C/hA3F. Here, we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein.

Functional characterization of Vif proteins from HIV-1 infected patients with different APOBEC3G haplotypes.

The human cytidine deaminase APOBEC3G (A3G) potently restricts HIV-1 but the virus, in turn, expresses a Vif protein which degrades A3G. A natural A3G-H186R variant, common in African populations, has been associated with a more rapid AIDS disease progression, but the underlying mechanism remains unknown. We hypothesized that differences in HIV-1 Vif activity towards A3G wild type and A3G-H186R contribute to the distinct clinical AIDS manifestation. Vif variants were cloned from plasma samples of 26 South African HIV-1 subtype C infected patients, which either express wild type A3G or A3G-H186R. The Vif alleles were assessed for their ability to counteract A3G variants using western blot and single-cycle infectivity assays. We obtained a total of 392 Vif sequences which displayed an amino acid sequence difference of 6.2-19.2% between patients. The intrapatient Vif diversities from patient groups A3G, A3G and A3G were similar. Vif variants obtained from patients expressing A3G and A3G were capable of counteracting both A3G variants with similar efficiency. However, the antiviral activity of A3G-H186R was significantly reduced in both the presence and absence of Vif, indicating that the A3G-H186R variant intrinsically exerts less antiviral activity. A3G wild type and A3G-H186R are equally susceptible to counteraction by Vif, regardless of whether the Vif variant was obtained from A3G and A3G patients. However, the A3G-H186R variant intrinsically displayed lower antiviral activity, which could explain the higher plasma viral loads and accelerated disease progression reported for patients expressing A3G.

The effect of HIV-1 Vif polymorphisms on A3G anti-viral activity in an in vivo mouse model.

Humans encode seven APOBEC3 proteins (A–H), with A3G, 3F and 3H as the major factors restricting HIV-1 replication. HIV-1, however, encodes Vif, which counteracts A3 proteins by chaperoning them to the proteasome where they are degraded. Vif polymorphisms found in HIV-1s isolated from infected patients have varying anti-A3G potency when assayed in vitro, but there are few studies demonstrating this in in vivo models. Here, we created Friend murine leukemia viruses encoding vif alleles that were previously shown to differentially neutralize A3G in cell culture or that were originally found in primary HIV-1 isolates. Infection of transgenic mice expressing different levels of human A3G showed that these naturally occurring Vif variants differed in their ability to counteract A3G during in vivo infection, although the effects on viral replication were not identical to those seen in cultured cells. We also found that the polymorphic Vifs that attenuated viral replication reverted to wild type only in A3G transgenic mice. Finally, we found that the level of A3G-mediated deamination was inversely correlated with the level of viral replication. This animal model should be useful for studying the functional significance of naturally occurring vif polymorphisms, as well as viral evolution in the presence of A3G.

Identification of the HIV-1 Vif and Human APOBEC3G Protein Interface.

Human cells express natural antiviral proteins, such as APOBEC3G (A3G), that potently restrict HIV replication. As a counter-defense, HIV encodes the accessory protein Vif, which binds A3G and mediates its proteasomal degradation. Our structural knowledge on how Vif and A3G interact is limited, because a co-structure is not available. We identified specific points of contact between Vif and A3G by using functional assays with full-length A3G, patient-derived Vif variants, and HIV forced evolution. These anchor points were used to model and validate the Vif-A3G interface. The resultant co-structure model shows that the negatively charged β4-α4 A3G loop, which contains primate-specific variation, is the core Vif binding site and forms extensive interactions with a positively charged pocket in HIV Vif. Our data present a functional map of this viral-host interface and open avenues for targeted approaches to block HIV replication by obstructing the Vif-A3G interaction.

Genetic and functional characterization of HIV-1 Vif on APOBEC3G degradation: First report of emergence of B/C recombinants from North India.

HIV-1 is characterized by high genetic heterogeneity which is a challenge for developing therapeutics. Therefore, 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. The functional domains, 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, suggesting its role in HIV-1 pathogenesis. This is the first report from North India showcasing genetic variations and novel polymorphisms in Vif gene. Subtype C is prevalent in India, 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.

Determinants of efficient degradation of APOBEC3 restriction factors by HIV-1 Vif.

The APOBEC3 deoxycytidine deaminases can restrict the replication of HIV-1 in cell culture to differing degrees. The effects of APOBEC3 enzymes are largely suppressed by HIV-1 Vif that interacts with host proteins to form a Cullin5-Ring E3 ubiquitin ligase that induces (48)K-linked polyubiquitination (poly-Ub) and proteasomal degradation of APOBEC3 enzymes. Vif variants have differing abilities to induce degradation of APOBEC3 enzymes and the underlying biochemical mechanisms for these differences is not fully understood. We hypothesized that by characterizing the interaction of multiple APOBEC3 enzymes and Vif variants we could identify common features that resulted in Vif-mediated degradation and further define the determinants required for efficient Vif-mediated degradation of APOBEC3 enzymes. We used Vifs from HIV-1 NL4-3 (IIIB) and HXB2 to characterize their induced degradation of and interaction with APOBEC3G, APOBEC3G D128K, APOBEC3H, and APOBEC3B in 293T cells. We quantified the APOBEC3G-Vif and APOBEC3H-Vif interaction strengths in vitro using rotational anisotropy. Our biochemical and cellular analyses of the interactions support a model in which the degradation efficiency of VifIIIB and VifHXB2 correlated with both the binding strength of the APOBEC3-Vif interaction and the APOBEC3-Vif interface, which differs for APOBEC3G and APOBEC3H. Notably, Vif bound to APOBEC3H and APOBEC3B in the natural absence of Vif-induced degradation and the interaction resulted in (63)K-linked poly-Ub of APOBEC3H and APOBEC3B, demonstrating additional functionality of the APOBEC3-Vif interaction apart from induction of proteasomal degradation. APOBEC3 enzymes can potently restrict the replication of HIV-1 in the absence of HIV-1 Vif. Vif suppresses APOBEC3 action by inducing their degradation through a direct interaction with APOBEC3 enzymes and other host proteins. Vif variants from different HIV-1 strains have different effects on APOBEC3 enzymes. We used differing Vif degradation capacities of two Vif variants and various APOBEC3 enzymes with differential sensitivities to Vif to delineate determinants of the APOBEC3-Vif interaction that are required for inducing efficient degradation. Using a combined biochemical and cellular approach we identified that the strength of the APOBEC3-Vif binding interaction and the APOBEC3-Vif interface are determinants for degradation efficiency. Our results highlight the importance of using Vif variants with different degradation potential when delineating mechanisms of Vif-induced APOBEC3 degradation and identify features important for consideration in the development of HIV-1 therapies that disrupt the APOBEC3-Vif interaction.

Suppression of APOBEC3-mediated restriction of HIV-1 by Vif.

The APOBEC3 restriction factors are a family of deoxycytidine deaminases that are able to suppress replication of viruses with a single-stranded DNA intermediate by inducing mutagenesis and functional inactivation of the virus. Of the seven human APOBEC3 enzymes, only APOBEC3-D, -F, -G, and -H appear relevant to restriction of HIV-1 in CD4+ T cells and will be the focus of this review. The restriction of HIV-1 occurs most potently in the absence of HIV-1 Vif that induces polyubiquitination and degradation of APOBEC3 enzymes through the proteasome pathway. To restrict HIV-1, APOBEC3 enzymes must be encapsidated into budding virions. Upon infection of the target cell during reverse transcription of the HIV-1 RNA into (-)DNA, APOBEC3 enzymes deaminate cytosines to form uracils in single-stranded (-)DNA regions. Upon replication of the (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite to the uracils thereby inducing C/G to T/A mutations that can functionally inactivate HIV-1. APOBEC3G is the most studied APOBEC3 enzyme and it is known that Vif attempts to thwart APOBEC3 function not only by inducing its proteasomal degradation but also by several degradation-independent mechanisms, such as inhibiting APOBEC3G virion encapsidation, mRNA translation, and for those APOBEC3G molecules that still become virion encapsidated, Vif can inhibit APOBEC3G mutagenic activity. Although most Vif variants can induce efficient degradation of APOBEC3-D, -F, and -G, there appears to be differential sensitivity to Vif-mediated degradation for APOBEC3H. This review examines APOBEC3-mediated HIV restriction mechanisms, how Vif acts as a substrate receptor for a Cullin5 ubiquitin ligase complex to induce degradation of APOBEC3s, and the determinants and functional consequences of the APOBEC3 and Vif interaction from a biological and biochemical perspective.

Vif Proteins from Diverse Primate Lentiviral Lineages Use the Same Binding Site in APOBEC3G

APOBEC3G (A3G) is a cytidine deaminase that restricts human immunodeficiency virus type 1 (HIV-1) and other lentiviruses. Most of these viruses encode a Vif protein that directly binds A3G and leads to its proteasomal degradation. Both Vif proteins of HIV-1 and African green monkey simian immunodeficiency virus (SIVagm) bind residue 128 of A3G. However, this position does not control the A3G degradation by Vif variants derived from HIV-2 and SIVmac, which both originated from SIV of sooty mangabey monkeys (SIVsmm), suggesting that the A3G binding site for Vif proteins of the SIVsmm/HIV-2 lineage differs from that of HIV-1. To map the SIVsmm Vif binding site of A3G, we performed immunoprecipitations of individual A3G domains, Vif/A3G degradation assays and a detailed mutational analysis of human A3G. We show that A3G residue 129, but not the adjacent position 128, confers susceptibility to degradation by SIVsmm Vif. An artificial A3G mutant, the P129D mutant, was resistant to degradation by diverse Vifs from HIV-1, HIV-2, SIVagm, and chimpanzee SIV (SIVcpz), suggesting a conserved lentiviral Vif binding site. Gorilla A3G naturally contains a glutamine (Q) at position 129, which makes its A3G resistant to Vifs from diverse lineages. We speculate that gorilla A3G serves as a barrier against SIVcpz strains. In summary, we show that Vif proteins from distinct lineages bind to the same A3G loop, which includes positions 128 and 129. The multiple adaptations within this loop among diverse primates underscore the importance of counteracting A3G in lentiviral evolution.

A Naturally Occurring Vif Mutant (I107T) Attenuates Anti-APOBEC3G Activity and HIV-1 Replication

The human immunodeficiency virus type 1 (HIV-1) Vif protein counteracts the antiviral activity of the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) family of proteins by targeting the proteins for degradation through the ubiquitin–proteasome pathway. Previous mutagenic studies have shown that multiple domains of Vif are required for interacting with APOBEC3G proteins and the proteasome pathway. However, very few mutagenesis and functional analyses of patient-derived Vif proteins have been conducted. In this study, we amplified and cloned the HIV-1 vif genes from the peripheral blood mononuclear cells (PBMCs) of five HIV-1-infected individuals in Nairobi and further tested the impact of the genes on anti-A3G activity and HIV-1 replication. The gene sequence analysis revealed high genetic variation of vif genes from different HIV-1-infected individuals. Interestingly, the Vif proteins derived from two of the three long-term survivors (LTSs) displayed a significantly impaired ability to mediate the degradation of A3G. In particular, a single amino acid change (I107T) in one of the non-functional LTS Vif variants, which has not been previously identified in the Los Alamos databases of vif sequences, was found to be responsible for the lack of anti-A3G activity. Further study demonstrated that HIV-1 carrying an I107T Vif mutation displayed significantly reduced fitness in A3G+ T cells and PBMCs. Moreover, co-infecting A3G+ T cells with both the wild-type and I107T Vif viruses resulted in decreased viral replication. Overall, the results of this study indicate that the HIV-1 Vif residue I107 is important for its anti-APOBEC3G activity and viral replication, which may have implications for viral fitness in vivo.

APOBEC3-Mediated Editing in HIV Type 1 from Pediatric Patients and Its Association withAPOBEC3G/CUL5Polymorphisms and Vif Variability

The APOBEC3 proteins are cytidine deaminases that can introduce G→A mutations in the HIV-1 plus DNA strand. This editing process may inhibit virus replication through lethal mutagenesis (hypermutation), but could also contribute to viral diversification leading to the emergence of escape forms. The HIV-1 Vif protein has the capacity to counteract APOBEC3 factors by recruiting a CUL5-based ubiquitin ligase complex that determines their proteasomal degradation. In this work, we analyzed the APOBEC3-mediated editing in proviral HIV-1 from perinatally infected children (n=93) in order to explore its association with polymorphisms of APOBEC3G and CUL5 genes (APOBEC3G H186R, APOBEC3G C40693T, and CUL5 SNP6), the Vif protein variability, and also the time to AIDS development. To calculate the level of editing, we have developed an index exploiting the properties of a region within the HIV-1 pol gene that includes the central polypurine tract (cPPT). We detected a reduced editing associated with the CUL5 SNP6 minor allele and also with certain Vif variants (mutations at sites 46, 122, and 160), although we found no evidence supporting an impact of APOBEC3 activity on disease progression. Thus, our findings suggest that APOBEC3-mediated editing of HIV-1 could be modulated by host and virus genetic characteristics in the context of pediatric infection.

The Activity Spectrum of Vif from Multiple HIV-1 Subtypes against APOBEC3G, APOBEC3F, and APOBEC3H

The APOBEC3 family comprises seven cytidine deaminases (APOBEC3A [A3A] to A3H), which are expressed to various degrees in HIV-1 susceptible cells. The HIV-1 Vif protein counteracts APOBEC3 restriction by mediating its degradation by the proteasome. We hypothesized that Vif proteins from various HIV-1 subtypes differ in their abilities to counteract different APOBEC3 proteins. Seventeen Vif alleles from seven HIV-1 subtypes were tested for their abilities to degrade and counteract A3G, A3F, and A3H haplotype II (hapII). We show that most Vif alleles neutralize A3G and A3F efficiently but display differences with respect to the inhibition of A3H hapII. The majority of non-subtype B Vif alleles tested presented some activity against A3H hapII, with two subtype F Vif variants being highly effective in counteracting A3H hapII. The residues required for activity were mapped to two residues in the amino-terminal region of Vif (positions 39F and 48H). Coimmunoprecipitations showed that these two amino acids were necessary for association of Vif with A3H hapII. These findings suggest that the A3H hapII binding site in Vif is distinct from the regions important for A3G and A3F recognition and that it requires specific amino acids at positions 39 and 48. The differential Vif activity spectra, especially against A3H hapII, suggest adaptation to APOBEC3 repertoires representative of different human ancestries. Phenotypic assessment of anti-APOBEC3 activity of Vif variants against several cytidine deaminases will help reveal the requirement for successful replication in vivo and ultimately point to interventions targeting the Vif-APOBEC3 interface.

Effect of HIV-1 Vif variability on progression to pediatric AIDS and its association with APOBEC3G and CUL5 polymorphisms

The APOBEC3G protein is a restriction factor that can inhibit the replication of HIV-1. The virus has the capacity to counteract this antiviral activity through the expression of the Vif accessory protein, which recruits a CUL5-based ubiquitin ligase complex that determines APOBEC3G proteasomal degradation. In this work we evaluated in a large pediatric cohort (i) whether single nucleotide polymorphisms of APOBEC3G and CUL5 genes (APOBEC3G H186R, APOBEC3G C40693T and CUL5 SNP6) can alter the risk of HIV-1 vertical transmission and/or the rate of progression to AIDS, (ii) the effect of HIV-1 Vif variants on the clinical course of disease, and (iii) whether the patient genotype for the studied polymorphisms could have an impact on Vif characteristics.We found no effect of the studied APOBEC3G or CUL5 genetic variants on vertical transmission or progression to pediatric AIDS. However, we detected an association of certain Vif alterations (a one amino acid insertion at position 61 and the substitutions A62D/N/S and Q136P) with an accelerated AIDS outcome. Additionally, we observed that the APOBEC3G C40693T and CUL5 SNP6 minor alleles were correlated with substitutions in Vif motifs that are involved in the interaction with APOBEC3G and CUL5 proteins, respectively. Our results suggest that Vif alterations may contribute to a rapid AIDS onset and that Vif variability could be influenced by APOBEC3G and CUL5 polymorphisms in children.

The Range of Human APOBEC3H Sensitivity to Lentiviral Vif Proteins

The APOBEC3H gene is polymorphic in humans, with four major population-dependent haplotypes that encode proteins with different levels of antiviral activity. Haplotype II, present most frequently in African populations, encodes the most stable protein and is most active against human immunodeficiency virus type 1 (HIV-1). In contrast to human APOBEC3G, which can be completely counteracted by HIV-1 Vif, the protein encoded by APOBEC3H haplotype II is only partially sensitive to Vif, while the protein encoded by APOBEC3H haplotype I is completely resistant to HIV-1 Vif. We mapped a residue on APOBEC3H that determines this partial Vif sensitivity. However, it is unclear how HIV-1 can replicate in vivo without the ability to neutralize APOBEC3H antiviral activity. In order to directly address this question, we cloned vif genes from HIV-1-infected individuals with different APOBEC3H genotypes and tested them for their ability to inhibit human APOBEC3H. We found that while the APOBEC3H genotype of infected individuals significantly influences the activity of Vif encoded by their virus, none of the Vif variants tested can completely neutralize APOBEC3H as well as they neutralize APOBEC3G. Consistent with this genetic result, APOBEC3H protein expression in human peripheral blood mononuclear cells was below our limit of detection using newly developed antibodies against the endogenous protein. These results demonstrate that human APOBEC3H is not as strong of a selective force for current HIV-1 infections as human APOBEC3G.

APOBEC3G induces a hypermutation gradient: purifying selection at multiple steps during HIV-1 replication results in levels of G-to-A mutations that are high in DNA, intermediate in cellular viral RNA, and low in virion RNA

BackgroundNaturally occurring Vif variants that are unable to inhibit the host restriction factor APOBEC3G (A3G) have been isolated from infected individuals. A3G can potentially induce G-to-A hypermutation in these viruses, and hypermutation could contribute to genetic variation in HIV-1 populations through recombination between hypermutant and wild-type genomes. Thus, hypermutation could contribute to the generation of immune escape and drug resistant variants, but the genetic contribution of hypermutation to the viral evolutionary potential is poorly understood. In addition, the mechanisms by which these viruses persist in the host despite the presence of A3G remain unknown.ResultsTo address these questions, we generated a replication-competent HIV-1 Vif mutant in which the A3G-binding residues of Vif, Y40RHHY44, were substituted with five alanines. As expected, the mutant was severely defective in an A3G-expressing T cell line and exhibited a significant delay in replication kinetics. Analysis of viral DNA showed the expected high level of G-to-A hypermutation; however, we found substantially reduced levels of G-to-A hypermutation in intracellular viral RNA (cRNA), and the levels of G-to-A mutations in virion RNA (vRNA) were even further reduced. The frequencies of hypermutation in DNA, cRNA, and vRNA were 0.73%, 0.12%, and 0.05% of the nucleotides sequenced, indicating a gradient of hypermutation. Additionally, genomes containing start codon mutations and early termination codons within gag were isolated from the vRNA.ConclusionThese results suggest that sublethal levels of hypermutation coupled with purifying selection at multiple steps during the early phase of viral replication lead to the packaging of largely unmutated genomes, providing a mechanism by which mutant Vif variants can persist in infected individuals. The persistence of genomes containing mutated gag genes despite this selection pressure indicates that dual infection and complementation can result in the packaging of hypermutated genomes which, through recombination with wild-type genomes, could increase viral genetic variation and contribute to evolution.