A single amino acid determinant governs the species-specific sensitivity of APOBEC3G to Vif action

APOBEC3G (also known as CEM15) is an innate intracellular antiretroviral factor that is counteracted by the Vif protein of lentiviruses. While APOBEC3G orthologues from several species are active against a broad range of retroviruses, given Vif proteins have a narrow spectrum of activity. For instance, HIV-1 Vif efficiently blocks APOBEC3G from human but not African green monkey (AGM), whereas the reverse is observed with SIV(AGM) Vif. Here, we demonstrate that a single amino acid at position 128 of human and AGM APOBEC3G governs the virus-specific sensitivity of these proteins to Vif-mediated inhibition. Furthermore, we show that this phenotype correlates with the ability of Vif to bind APOBEC3G and interfere with its incorporation into virions. These results shed light on an important determinant of the tropism of primate lentiviruses.     

Identification of two distinct human immunodeficiency virus type 1 Vif determinants critical for interactions with human APOBEC3G and APOBEC3F

Human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) inhibit replication of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif overcomes these host restriction factors by binding to them and inducing their proteasomal degradation. The Vif-A3G and Vif-A3F interactions are attractive targets for antiviral drug development because inhibiting the interactions could allow the host defense mechanism to control HIV-1 replication. It was recently reported that the Vif amino acids D(14)RMR(17) are important for functional interaction and degradation of the previously identified Vif-resistant mutant of A3G (D128K-A3G). However, the Vif determinants important for functional interaction with A3G and A3F have not been fully characterized. To identify these determinants, we performed an extensive mutational analysis of HIV-1 Vif. Our analysis revealed two distinct Vif determinants, amino acids Y(40)RHHY(44) and D(14)RMR(17), which are essential for binding to A3G and A3F, respectively. Interestingly, mutation of the A3G-binding region increased Vif's ability to suppress A3F. Vif binding to D128K-A3G was also dependent on the Y(40)RHHY(44) region but not the D(14)RMR(17) region. Consistent with previous observations, subsequent neutralization of the D128K-A3G antiviral activity required substitution of Vif determinant D(14)RMR(17) with SEMQ, similar to the SERQ amino acids in simian immunodeficiency virus SIV(AGM) Vif, which is capable of neutralizing D128K-A3G. These studies are the first to clearly identify two distinct regions of Vif that are critical for independent interactions with A3G and A3F. Pharmacological interference with the Vif-A3G or Vif-A3F interactions could result in potent inhibition of HIV-1 replication by the APOBEC3 proteins.     

Further investigation of simian immunodeficiency virus Vif function in human cells

Primate lentivirus Vif proteins function by suppressing the antiviral activity of the cell-encoded apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) proteins APOBEC3G and APOBEC3F. It has been hypothesized that species-specific susceptibilities of APOBEC proteins to Vif proteins may help govern the transmission of primate lentiviruses to new host species. Consistent with this view and with previous results, we report that the Vif proteins of several diverse simian immunodeficiency viruses (SIVs) that are not known to infect humans are not effective inhibitors of human APOBEC3G or APOBEC3F when assessed in transient-transfection experiments. Unexpectedly, this lack of SIV Vif function did not prevent the replication of two vif-deficient SIVs (SIVtan and SIVmnd1; isolated from tantalus monkeys and mandrills, respectively) in a human T-cell line, HUT78, that expresses both APOBEC 3G and APOBEC3F, a finding which demonstrates that some SIVs are partially resistant to the antiretroviral effects of these enzymes irrespective of Vif function. Additional virus replication studies also revealed that the Vif protein of SIVtan is, in fact, active in human T cells, as it substantially enhanced the replication of its cognate virus and human immunodeficiency virus type 1. In sum, we now consider it improbable that species-specific restrictions to SIV Vif function can explain the lack of human infection with certain SIVs. Instead, our data reveal that the species-specific modulation of Vif function is more complex than previously envisioned and that additional (as-yet-unidentified) viral or host factors may be involved in regulating this dynamic interaction between host and pathogen.     

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.     

Influence of primate lentiviral Vif and proteasome inhibitors on human immunodeficiency virus type 1 virion packaging of APOBEC3G

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral evasion of the host antiviral protein APOBEC3G, also known as CEM15. Vif mutant but not wild-type HIV-1 viruses produced in the presence of APOBEC3G have been shown to undergo hypermutations in newly synthesized viral DNA upon infection of target cells, presumably resulting from C-to-U modification during minus-strand viral DNA synthesis. We now report that HIV-1 Vif could induce rapid degradation of human APOBEC3G that was blocked by the proteasome inhibitor MG132. The efficiency of Vif-induced downregulation of APOBEC3G expression depended on the level of Vif expression. A single amino acid substitution in the conserved SLQXLA motif reduced Vif function. Vif proteins from distantly related primate lentiviruses such as SIVagm were unable to suppress the antiviral activity of human APOBEC3G or the packaging of APOBEC3G into HIV-1 Vif mutant virions, due to a lack of interaction with human APOBEC3G. In the presence of the proteasome inhibitor MG132, virion-associated Vif increased dramatically. However, increased virion packaging of Vif did not prevent virion packaging of APOBEC3G when proteasome function was impaired, and the infectivity of these virions was significantly reduced. These results suggest that Vif function is required during virus assembly to remove APOBEC3G from packaging into released virions. Once packaged, virion-associated Vif could not efficiently block the antiviral activity of APOBEC3G.     

Wide range of viral load in healthy african green monkeys naturally infected with simian immunodeficiency virus

The distribution and levels of simian immunodeficiency virus (SIV) in tissues and plasma were assessed in naturally infected African green monkeys (AGM) of the vervet subspecies (Chlorocebus pygerythrus) by limiting-dilution coculture, quantitative PCR for viral DNA and RNA, and in situ hybridization for SIV expression in tissues. A wide range of SIV RNA levels in plasma was observed among these animals (<1,000 to 800,000 copies per ml), and the levels appeared to be stable over long periods of time. The relative numbers of SIV-expressing cells in tissues of two monkeys correlated with the extent of plasma viremia. SIV expression was observed in lymphoid tissues and was not associated with immunopathology. Virus-expressing cells were observed in the lamina propria and lymphoid tissue of the gastrointestinal tract, as well as within alveolar macrophages in the lung tissue of one AGM. The range of plasma viremia in naturally infected AGM was greater than that reported in naturally infected sooty mangabeys. However, the degree of viremia in some AGM was similar to that observed during progression to AIDS in human immunodeficiency virus-infected individuals. Therefore, containment of viremia is an unlikely explanation for the lack of pathogenicity of SIVagm in its natural host species, AGM.     

Production of infectious SIVagm from human cells requires functional inactivation but not viral exclusion of human APOBEC3G

The virus infectivity factor (Vif) is a protein encoded by most primate lentiviruses. Recent evidence suggests that HIV-1 Vif reduces the intracellular levels of the host cytidine deaminase APOBEC3G (Apo3G) and inhibits its packaging into virions. These functions of Vif are thought to be species-specific. Accordingly, HIV-1 Vif can target only human Apo3G (hApo3G), whereas, African green monkey simian immunodeficiency virus (SIVagm) Vif can inhibit African green monkey but not human Apo3G. Consistent with this, we found that SIVagm Vif does not affect the stability of exogenously and endogenously expressed hApo3G and does not prevent packaging of exogenous and endogenous hApo3G into SIVagm virions. Nevertheless, SIVagm Vif supported spreading infection of SIVagm virus in the hApo3G-positive human A3.01 T cell line and rescued infectivity of viruses produced from Apo3G-expressing HeLa cells. Sequence analysis verified that SIVagm Vif inhibited the accumulation of hApo3G-induced mutations, suggesting that SIVagm Vif is indeed active in human cells. Our data suggest that SIVagm Vif can inhibit hApo3G activity without inducing its intracellular degradation or preventing its packaging into virions.     

Primate lentiviral Vpx commandeers DDB1 to counteract a macrophage restriction

Primate lentiviruses encode four "accessory proteins" including Vif, Vpu, Nef, and Vpr/Vpx. Vif and Vpu counteract the antiviral effects of cellular restrictions to early and late steps in the viral replication cycle. We present evidence that the Vpx proteins of HIV-2/SIV(SM) promote virus infection by antagonizing an antiviral restriction in macrophages. Fusion of macrophages in which Vpx was essential for virus infection, with COS cells in which Vpx was dispensable for virus infection, generated heterokaryons that supported infection by wild-type SIV but not Vpx-deleted SIV. The restriction potently antagonized infection of macrophages by HIV-1, and expression of Vpx in macrophages in trans overcame the restriction to HIV-1 and SIV infection. Vpx was ubiquitylated and both ubiquitylation and the proteasome regulated the activity of Vpx. The ability of Vpx to counteract the restriction to HIV-1 and SIV infection was dependent upon the HIV-1 Vpr interacting protein, damaged DNA binding protein 1 (DDB1), and DDB1 partially substituted for Vpx when fused to Vpr. Our results indicate that macrophage harbor a potent antiviral restriction and that primate lentiviruses have evolved Vpx to counteract this restriction.     

The Role of the Antiviral APOBEC3 Gene Family in Protecting Chimpanzees against Lentiviruses from Monkeys

Cross-species transmissions of viruses from animals to humans are at the origin of major human pathogenic viruses. While the role of ecological and epidemiological factors in the emergence of new pathogens is well documented, the importance of host factors is often unknown. Chimpanzees are the closest relatives of humans and the animal reservoir at the origin of the human AIDS pandemic. However, despite being regularly exposed to monkey lentiviruses through hunting, chimpanzees are naturally infected by only a single simian immunodeficiency virus, SIVcpz. Here, we asked why chimpanzees appear to be protected against the successful emergence of other SIVs. In particular, we investigated the role of the chimpanzee APOBEC3 genes in providing a barrier to infection by most monkey lentiviruses. We found that most SIV Vifs, including Vif from SIVwrc infecting western-red colobus, the chimpanzee’s main monkey prey in West Africa, could not antagonize chimpanzee APOBEC3G. Moreover, chimpanzee APOBEC3D, as well as APOBEC3F and APOBEC3H, provided additional protection against SIV Vif antagonism. Consequently, lentiviral replication in primary chimpanzee CD4⁺ T cells was dependent on the presence of a lentiviral vif gene that could antagonize chimpanzee APOBEC3s. Finally, by identifying and functionally characterizing several APOBEC3 gene polymorphisms in both common chimpanzees and bonobos, we found that these ape populations encode APOBEC3 proteins that are uniformly resistant to antagonism by monkey lentiviruses.     

Conservation and host specificity of Vpr-mediated cell cycle arrest suggest a fundamental role in primate lentivirus evolution and biology.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein prevents infected cells from passing through mitosis by arresting them in the G2 phase of the cell cycle. Vpr is conserved among all primate lentiviruses, suggesting an important role in the virus life cycle. Moreover, in this study we show that the ability to cause cell cycle arrest is also conserved in Vpr proteins from a wide variety of both tissue culture-passaged and uncultured human (HIV-1 and HIV-2), sooty mangabey (simian immunodeficiency virus SIV(SM)), African green monkey (SIV(AGM)), and Sykes' monkey (SIV(SYK)) isolates. However, this property is cell type specific and appears to depend on the particular primate species from which the cells are derived. SIV(AGM) and SIV(SYK) Vpr proteins are capable of arresting African green monkey cells but are completely inactive in human cells. By contrast, HIV-1, HIV-2, and SIV(SM) Vpr proteins function in both simian and human cell types, although SIV(SM) Vpr functions more efficiently in simian cells than it does in human cells. Neither differential protein stability nor subcellular localization explains the species-specific activities of these proteins. These results thus suggest that Vpr exerts its G2 arrest function by interacting with cellular factors that have evolved differently among the various primate species.     

Vif counteracts a cyclophilin A-imposed inhibition of simian immunodeficiency viruses in human cells

Vif is a primate lentiviral accessory protein that is crucial for viral infectivity. Vif counteracts the antiviral activity of host deaminases such as APOBEC3G and APOBEC3F. We now report a novel function of African green monkey simian immunodeficiency virus (SIVagm) Vif that promotes replication of SIVagm in human cells lacking detectable deaminase activity. We found that cyclophilin A (CypA) was excluded from wild-type SIV particles but was efficiently packaged into vif-deficient SIVagm virions. The presence of CypA in vif-defective SIVagm was correlated with reduced viral replication. Infection of CypA knockout Jurkat cells or treatment of Jurkat cells with cyclosporine A eliminated the Vif-sensitive inhibition and resulted in replication profiles that were similar for wild-type and vif-deficient SIVagm. Importantly, the inhibitory effect of CypA was restricted to virus-producing cells and was TRIM5alpha independent. The abilities of SIVagm Vif to inhibit encapsidation of CypA and to increase viral infectivity were shared by rhesus macaque SIV Vif and thus seem to be general properties of SIV Vif proteins. Exclusion of CypA from SIVagm particles was not associated with intracellular degradation, suggesting a mode of Vif action distinct from that proposed for APOBEC3G. This is the first report of a novel vif-sensitive antiviral activity of human CypA that may limit zoonotic transmission of SIV and the first demonstration of CypA encapsidation into a virus other than human immunodeficiency virus type 1.     

Simian immunodeficiency viruses from central and western Africa: evidence for a new species-specific lentivirus in tantalus monkeys.

Although up to 50% of African green monkeys (AGMs) are infected by simian immunodeficiency viruses (SIV) in their natural habitat, they remain asymptomatic carriers of these lentiviruses. They provide an attractive model to study not only the origin but also the link among genetic variation, host-virus adaptation, and pathogenicity of primate lentiviruses. SIVagm have been isolated from three species of AGM: the vervet (Cercopithecus pygerythrus), the grivet (Cercopithecus aethiops), and the sabaeus (Cercopithecus sabaeus) monkey. We studied four new SIVagm isolates from a fourth AGM species, the tantalus monkey (Cercopithecus tantalus), caught in the Central African Republic, and four new isolates from feral sabaeus monkeys from Senegal. Antigenic properties and partial env sequences were used to evaluate the diversity among these isolates. Alignment of env sequences in SIVagm isolated from tantalus and sabaeus monkeys permitted detailed mapping of the variable and conserved domains in the external glycoprotein. Genetic distances indicated that SIVagm isolates from tantalus monkeys are the most divergent among SIVagm in feral AGMs in Africa. The fact that AGMs are infected by four distinct lentiviruses, each specific for a single AGM species, supports the hypothesis of a coevolution of these viruses and their natural hosts and suggests that SIV transmission is a rare event among separated AGM species in the wild.     

Regulation of Apobec3F and human immunodeficiency virus type 1 Vif by Vif-Cul5-ElonB/C E3 ubiquitin ligase

The human cytidine deaminase Apobec3F (h-A3F), a protein related to the previously recognized antiviral factor Apobec3G (h-A3G), has antiviral activity against human immunodeficiency virus type 1 (HIV-1) that is suppressed by the viral protein Vif. The mechanism of HIV-1 Vif-mediated suppression of h-A3F is not fully understood. Here, we demonstrate that while h-A3F, like h-A3G, was able to suppress primate lentiviruses other than HIV-1 (simian immunodeficiency virus from African green monkeys [SIVagm] and Rhesus macaques [SIVmac]), the interaction between Vif proteins and h-A3F appeared to differ from that with h-A3G. H-A3F showed no change in its species specificity against HIV-1 or SIVagm Vif when a negatively charged amino acid was replaced with a lysine at position 128, a residue critical for h-A3G recognition by HIV-1 Vif. However, HIV-1 Vif, but not SIVagm Vif, was able to bind h-A3F and induce its polyubiquitination and degradation through the Cul5-containing E3 ubiquitin ligase. Interference with Cul5-E3 ligase function by depletion of Cul5, through RNA interference or overexpression of Cul5 mutants, blocked the ability of HIV-1 Vif to suppress h-A3F. A BC-box mutant of HIV-1 Vif that failed to recruit Cul5-E3 ligase but was still able to interact with h-A3F failed to suppress h-A3F. Interestingly, interference with Cul5-E3 ligase function or overexpression of h-A3F or h-A3G also increased the stability of HIV-1 Vif, suggesting that like the substrate molecules h-A3F and h-A3G, the substrate receptor protein Vif is itself also regulated by Cul5-E3 ligase. Our results indicate that Cul5-E3 ligase appears to be a common pathway hijacked by HIV-1 Vif to defeat both h-A3F and h-A3G. Developing inhibitors to disrupt the interaction between Vif and Cul5-E3 ligase could be therapeutically useful, allowing multiple host antiviral factors to suppress HIV-1.     

Conserved and non-conserved features of HIV-1 and SIVagm Vif mediated suppression of APOBEC3 cytidine deaminases

Human cytidine deaminase APOBEC3C (A3C) acts as a potent inhibitor of SIVagm and can be regulated by both HIV-1 and SIVagm Vif. The mechanism by which Vif suppresses A3C is unknown. In the present study, we demonstrate that both HIV-1 and SIVagm Vif can act in a proteasome-dependent manner to overcome A3C. SIVagm Vif requires the Cullin5-ElonginB-ElonginC E3 ubiquitin ligase for the degradation of A3C as well as the suppression of its antiviral activity. Mutation of a residue critical for the species-specific recognition of human or monkey A3G by HIV-1 Vif or SIVagm Vif in A3C had little effect on HIV-1 or SIVagm Vif-mediated degradation of A3C. Although the amino-terminal region of A3G was not important for Vif-mediated degradation, the corresponding region in A3C was critical. A3C mutants that were competent for Vif binding but resistant to Vif-mediated degradation were identified. These data suggest that primate lentiviral Vif molecules have evolved to recognize multiple host APOBEC3 proteins through distinct mechanisms. However, Cul5-E3 ubiquitin ligase appears to be a common pathway hijacked by HIV-1 and SIV Vif to defeat APOBEC3 proteins. Furthermore, Vif and APOBEC3 binding is not sufficient for target protein degradation indicating an important but uncharacterized Vif function.     

Rates of amino acid change in the envelope protein correlate with pathogenicity of primate lentiviruses

A spectrum of pathogenicity has been observed for primate lentiviruses in their natural hosts. For example, human immunodeficiency virus type 1 (HIV-1) is a potent etiologic agent for AIDS in man, whereas there is no evidence to date which indicates that simian immunodeficiency virus from African green monkeys (SIVAGM) causes immunodeficiency in AGM. We measured the relative rates of amino acid change, as the ratio of the number of nonsynonymous to synonymous (silent) nucleotide substitutions, for six primate lentiviruses evolving in their respective hosts. These rates for the external envelope glycoprotein (gp120) and gag coding sequences are 2–3 times higher for pathogenic HIV-1 and SIV..ac (macaque) than for minimally pathogenic SIVAGM and SIVsn,m (sooty mangabey), and intermediate for HIV-2. We speculate that the increased rates of nonsynonymous changes in gp120 and gag coding sequences are due to viral escape from immune surveillance and are indicative of higher immunogenicity of these proteins in their hosts. Based on these results and available experimental data, we conclude that there is a positive correlation between lentiviral pathogenicity and immunogenicity of the Env and Gag proteins in a given host. This hypothesis is consistent with recent data suggesting that immune system activation or autoimmunity induced by viral antigens may be important in the pathogenesis of AIDS.Correspondence to: E.G. Shpaer     

A challenge to the ancient origin of SIVagm based on African green monkey mitochondrial genomes

While the circumstances surrounding the origin and spread of HIV are becoming clearer, the particulars of the origin of simian immunodeficiency virus (SIV) are still unknown. Specifically, the age of SIV, whether it is an ancient or recent infection, has not been resolved. Although many instances of cross-species transmission of SIV have been documented, the similarity between the African green monkey (AGM) and SIVagm phylogenies has long been held as suggestive of ancient codivergence between SIVs and their primate hosts. Here, we present well-resolved phylogenies based on full-length AGM mitochondrial genomes and seven previously published SIVagm genomes; these allowed us to perform the first rigorous phylogenetic test to our knowledge of the hypothesis that SIVagm codiverged with the AGMs. Using the Shimodaira-Hasegawa test, we show that the AGM mitochondrial genomes and SIVagm did not evolve along the same topology. Furthermore, we demonstrate that the SIVagm topology can be explained by a pattern of west-to-east transmission of the virus across existing AGM geographic ranges. Using a relaxed molecular clock, we also provide a date for the most recent common ancestor of the AGMs at approximately 3 million years ago. This study substantially weakens the theory of ancient SIV infection followed by codivergence with its primate hosts.