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J Virol. Aug 2010; 84(16): 8193–8201.
Published online Jun 2, 2010. doi:  10.1128/JVI.00685-10
PMCID: PMC2916508

Lentiviral Vif Degrades the APOBEC3Z3/APOBEC3H Protein of Its Mammalian Host and Is Capable of Cross-Species Activity[down-pointing small open triangle]

Abstract

All lentiviruses except equine infectious anemia virus (EIAV) use the small accessory protein Vif to counteract the restriction activity of the relevant APOBEC3 (A3) proteins of their host species. Prior studies have suggested that the Vif-A3 interaction is species specific. Here, using the APOBEC3H (Z3)-type proteins from five distinct mammals, we report that this is generally not the case: some lentiviral Vif proteins are capable of triggering the degradation of both the A3Z3-type protein of their normal host species and those of several other mammals. For instance, SIVmac Vif can mediate the degradation of the human, macaque, and cow A3Z3-type proteins but not of the sheep or cat A3Z3-type proteins. Maedi-visna virus (MVV) Vif is similarly promiscuous, degrading not only sheep A3Z3 but also the A3Z3-type proteins of humans, macaques, cows, and cats. In contrast to the neutralization capacity of these Vif proteins, human immunodeficiency virus (HIV), bovine immunodeficiency virus (BIV), and feline immunodeficiency virus (FIV) Vif appear specific to the A3Z3-type protein of their hosts. We conclude, first, that the Vif-A3Z3 interaction can be promiscuous and, second, despite this tendency, that each lentiviral Vif protein is optimized to degrade the A3Z3 protein of its mammalian host. Our results thereby suggest that the Vif-A3Z3 interaction is relevant to lentivirus biology.

Lentiviruses are a unique class of complex retroviruses that encode a variety of accessory proteins in addition to the required Gag, Pol, and Env proteins. The archetypal lentivirus, human immunodeficiency virus type 1 (HIV-1), infects humans, but other members include simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), maedi-visna virus (MVV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and feline immunodeficiency virus (FIV), which infect monkeys, cattle, sheep, goats, horses, and cats, respectively. The HIV-1 accessory protein viral infectivity factor (Vif) has been extensively studied because of its essential function in inhibiting the cellular antiretroviral human APOBEC3G (A3G) protein (43). HIV-1 Vif binds to human A3G (and other A3 proteins) and serves as an adaptor to link it to an ELOC-based E3 ubiquitin ligase complex (30, 51, 52). A3G is then polyubiquitinated and degraded by the cellular proteasome (7, 15, 29, 30, 43, 46, 52).

Due to the potential therapeutic value of disrupting this host-pathogen interaction, a significant amount of work has been invested in defining the important contact residues between A3G and HIV-1 Vif. Primate A3G homologs have been useful tools in this effort, as many fail to be neutralized by HIV-1 Vif despite a relatively high degree of sequence similarity. For example, while HIV-1 Vif effectively neutralizes human A3G, it does not neutralize African green monkey A3G or rhesus macaque A3G despite 77% and 75% identity, respectively (4, 26, 27, 41, 51). The differential capacity of the HIV-1 and SIVagm Vif proteins to degrade the A3G proteins of their hosts led to demonstrations that residue 128 is a key determinant: D128 made each A3G protein susceptible to HIV-1 Vif and K128 made each A3G protein susceptible to SIVagm Vif (4, 26, 41, 51). This apparent on/off switch led to the prevailing model that the Vif-A3 interaction is species specific. However, even early data sets showed at least two hints that the story was more complex. First, the identity of the A3G residue 128 (K or D) does not diminish the interaction with the Vif proteins of SIVmac or HIV-2 (41, 51). Second, SIVmac Vif was shown to potently counteract the A3G proteins from rhesus macaque (as expected) but also those from human, African green monkey, and chimpanzee (27). Therefore, the implication from these studies is that the full nature of the A3-Vif interaction has yet to be elucidated.

Although A3G has clearly served as the prototype for understanding the A3-Vif interaction, a growing number of studies indicate that other A3s are also capable of restricting lentivirus replication and interacting with Vif. A3G is one of seven human A3 proteins (A3A to -H) encoded in tandem on chromosome 22 (7, 16, 49). All but A3A have been implicated in the restriction of HIV-1 replication (reviewed in references 1, 10, and 45). For instance, human A3H has been shown to restrict HIV-1 replication and is susceptible to degradation by HIV-1 Vif (8, 37, 47). A3H is a Z3-type DNA deaminase characterized by a conserved threonine and a valine, in addition to the canonical H-x1-E-x23-28-C-x2-4-C zinc-coordinating motif (23). The Z3-type deaminase is unique in that only one copy exists in all mammals whose genomes have been sequenced. It is encoded by a five-exon gene located at the distal end of each mammal's A3 locus (adjacent to CBX7). Additional observations suggest that the Z3-type deaminases appear to have the capacity to restrict the Vif-deficient lentiviruses of their hosts. For example, African green monkey A3H can restrict the replication of SIVagm and is susceptible to degradation by SIVagm Vif, and the cat A3Z3 can restrict the replication of FIV and is susceptible to degradation by FIV Vif (33, 37, 48).

Here, we take advantage of the fact that all sequenced mammals have a single A3Z3-type protein to test the hypothesis that these proteins are of general relevance to lentivirus restriction and to clarify the species-specific nature of the mammalian A3Z3/lentiviral Vif relationship. First, we ask if human, rhesus macaque, cow, sheep, and cat A3Z3-type proteins are all capable of retrovirus restriction. Second, we ask whether they are susceptible to Vif-mediated degradation in a host-specific manner. We show that each lentiviral Vif protein can indeed neutralize the Z3-type A3 protein of its host species. However, we were surprised to find that several of the Vif proteins, particularly SIVmac and MVV Vif, can neutralize a broad number of A3Z3 proteins irrespective of the species of origin and overall degree of similarity. These data indicate that the A3-Vif interaction is more promiscuous than previously appreciated. Such broad functional flexibility may be relevant to understanding past retroviral zoonoses and predicting potential future events. We conclude that the A3Z3-Vif interaction is conserved on a macroscopic level, consistent with an important role in viral replication and particularly in species like artiodactyls and felines with fewer A3 proteins.

MATERIALS AND METHODS

Sequence alignments.

Mammalian A3Z3 protein sequences were aligned and scored using the default matrix BLOSUM62 by protein BLAST (2, 14). The following A3Z3 sequences were compared and tested in this study: human A3H (gb EU861361), rhesus macaque A3H (gb DQ507277), cow A3Z3 (gb EU864536), sheep A3Z3 (gb EU864543), and cat A3Z3 (gb EU011792). Protein sequences for the lentiviral Vif proteins were aligned using T_coffee version 5.31 (35). All GenBank accession numbers for the lentiviral Vif protein sequences are listed in the “Vif expression plasmids” section below.

APOBEC3 expression plasmids.

The cow A3Z1, A3Z2, A3Z3, and A3Z2-Z3 and sheep A3Z3 genes have been described previously (17, 23). These cDNAs were subcloned from the pEGFP-N3 expression vector by being digested with KpnI and SalI and inserted into a similarly digested pcDNA3.1 eukaryotic expression vector with no stop codon. This expression vector added three consecutive hemagglutinin (HA) epitope tags to the C-terminal domain (CTD) of the encoded A3 protein. All of the remaining mammalian A3Z3 genes were also inserted into the pcDNA3.1 expression vector, and details are described below. The human A3H (haplotype II) and rhesus macaque A3H cDNAs were kindly provided by M. Emerman (37, 38). These A3H sequences contained an N-terminal domain (NTD) tag that was removed by PCR amplification with these primers: human, forward, 5′-NNN NGA GCT CGG TAC CAC CAT GGC TCT GTT AAC AGC CGA AAC-3′, and reverse, 5′-NNN GTC GAC TCC GGA CTG CTT TAT CCT CTC AAG CC-3′, and rhesus macaque, forward, 5′-NNN NGA GCT CGG TAC CAC CAT GGC TCT GCT AAC AGC-3′, and reverse, 5′-NNN GTC GAC TCC TCT TGA GTT GCG TAT TGA CGA TG-3′. The untagged human A3H (haplotype II) was amplified with the same forward primer and a reverse primer with a stop codon, 5′-NNN NNG TCG ACT CAG GAC TGC TTT ATC CTC TCA AGC CGT C-3′. To ensure the same sequence for the cat A3Z3 gene from reference 33, RNA was extracted from Crandell-Rees feline kidney (CRFK) cells and reverse transcribed and 3′ rapid amplification of cDNA ends (RACE) was performed on the cDNA as described previously (23). The following primers were used to amplify the cat A3Z3 gene without a stop codon: forward, 5′-NNN NGA GCT CAG GTA CCA CCA TGA ATC CAC TAC AGG AA-3′, and reverse, 5′-NNN NGT CGA CTT CAA GTT TCA AAT TTC TGA A-3′. The mouse A3Z2-Z3 and human A3G expression plasmids were described elsewhere (11, 13, 17).

Vif expression plasmids.

The lentiviral Vifs chosen for codon optimization (GenScript Corporation) match HIV-1IIIB (gb EU541617), SIVmac239 (gb AY588946), BIVBIM127 (gb M32690), MVV Icelandic strain 1514 (gb M60610), and FIVNSCU (gb M25381). All of the Vifs were amplified by PCR from the pUC57 vector (GenScript Corporation) and cloned into the pVR1012 vector (Vical Co.). The HIV-1LAI Vif in the pVR1012 vector was a generous gift from X. F. Yu, and it is not codon optimized. All Vifs were amplified by PCR and designed to add a c-myc sequence to the CTD, digested with NotI or SalI and BamHI, and cloned into the pVR1012 vector. The following primers were used for the lentiviral Vifs: HIV-1IIIB Vif, forward, 5′-CAC AAC AAG GTG GGC GCA GCG GCG TAC CTT GCA CTG GCC-3′, and reverse, 5′-GGA TCC CTA CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC GTG GCC ATT CAT TGT-3′; HIV-1LAI Vif, forward, 5′-GTC GAC GCC ACC ATG GAA AAC AGA TGG-3′, and reverse, 5′-NNN NGG ATC CCT ACA GAT CCT CTT CTG AGA TGA GTT TTT GTT CGT GTC CAT TCA TTG T-3′; SIVmac Vif, forward, 5′-AAG TAC CAG GTT CCT GCT GCG GCG TAT CTG GCA CTC AAA-3′, and reverse, 5′-GGA TCC CTA CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC GGC CAG GAT ACC CAG-3′; BIV Vif, forward, 5′-CTC TAC CCC ACG CCA CGC CGC GGC GCG GCT GGC AGC TCT G-3′, and reverse, 5′-GGA TCC CTA CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC AGG GTG TCC GCT CAG-3′; MVV Vif, forward, 5′-AAC ACT AAC CCC AGA GCC GCG GCG AGA CTT GCC CTG CTT-3′, and reverse, 5′-GGA TCC CTA CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC CTC AAA AAT GCT CTC-3′; and FIV Vif, forward, 5′-AAC AGC CCA CCA CAG GCC GCG GCG CGG CTG GCC ATG CTG-3′, and reverse, 5′-GGA TCC CTA CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC CAG GGA GCC AGA CCA-3′.

Vif mutants were constructed using standard site-directed mutagenesis protocols (Stratagene). The following primers were used on the codon-optimized lentiviral Vifs: HIV-1IIIB Vif (SLQ to AAA), forward, 5′-CAC AAC AAG GTG GGC GCA GCG GCG TAC CTT GCA CTG GCC-3′, and reverse, 5′-GGC CAG TGC AAG GTA CGC CGC TGC GCC CAC CTT GTT GTG-3′; SIVmac Vif (SLQ to AAA), forward, 5′-AAG TAC CAG GTT CCT GCT GCG GCG TAT CTG GCA CTC AAA-3′, and reverse, 5′-TTT GAG TGC CAG ATA CGC CGC AGC AGG AAC CTG GTA CTT-3′; BIV Vif (SLQ to AAA), forward, 5′-CTC TAC CCC ACG CCA CGC CGC GGC GCG GCT GGC AGC TCT G-3′, and reverse, 5′-CAG AGC TGC CAG CCG CGC CGC GGC GTG GCG TGG GGT AGA G-3′; MVV Vif (SLQ to AAA), forward, 5′-AAC ACT AAC CCC AGA GCC GCG GCG AGA CTT GCC CTG CTT-3′, and reverse, 5′-AAG CAG GGC AAG TCT CGC CGC GGC TCT GGG GTT AGT GTT-3′; and FIV Vif (TLQ to AAA), forward, 5′-AAC AGC CCA CCA CAG GCC GCG GCG CGG CTG GCC ATG CTG-3′, and reverse, 5′-CAG CAT GGC CAG CCG CGC CGC GGC CTG TGG TGG GCT GTT-3′. The following primers were used for HIV-1LAI Vif (SLQ to AAA): forward, 5′-GAC ATA ACA AGG TAG GAG CTG CTG CAT ACT TGG CAC TAG CA-3′, and reverse, 5′-TGC TAG TGC CAA GTA TGC AGC AGC TCC TAC CTT GTT ATG TC-3′.

HIV-GFP infectivity assays.

Human 293T cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 0.5% penicillin-streptomycin. At 50% confluence, cells were transfected (TransIt; Mirus) with 1 μg HIV-green fluorescent protein (GFP) cocktail [0.44 μg of CS-CG (32), 0.28 μg of pRK5/Pack1(Gag-Pol), 0.14 μg pRK5/Rev, 0.14 μg of pMDG (vesicular stomatitis virus G protein [VSV-G]-Env)], 100 ng of A3 or empty vector, and 20 ng of codon-optimized Vif or 400 ng of wild-type HIV-1LAI Vif with corresponding empty vector. The ratio of A3 to Vif constructs was based on whether or not the Vif was codon optimized. All experiments were performed in triplicate except where noted in the legends.

After 48 h, virus-containing supernatants were harvested and purified by centrifugation to remove any remaining producer cells. Producer cells were processed for Western analysis and GFP+ flow cytometry (Cell Lab Quanta SC-MPL; Beckman Coulter) to measure protein expression levels and transfection efficiencies, respectively. The purified supernatants were placed on fresh 293T target cells. After 72 h, target cells were harvested and infectivity (GFP) was measured by flow cytometry. Data were analyzed using FlowJo flow cytometry analysis software, version 8.7.1.

Western blotting of cell lysates and viral particles.

Producer cell lysates were harvested and pooled from triplicate experiments. Lysates and viral particle pellets were resuspended in 2× Laemmli sample buffer (25 mM Tris, pH 6.8, 8% glycerol, 0.8% SDS, 2% 2-mercaptoethanol, 0.02% bromophenol blue) and heated at 98°C degrees for 10 min, and a fraction of the sample was run on a 12% SDS-PAGE gel. Protein was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). Membranes with cell lysates were first probed with anti-c-myc (Sigma-Aldrich) for detection of Vif expression and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody and developed using a chemiluminescent HRP antibody detection reagent (HyGLO; Denville Scientific). Blots were then stripped (0.2 M glycine, 1.0% SDS, 1.0% Tween 20, pH 2.2), and this protocol was repeated with an anti-HA antibody (Covance) to detect A3 proteins and with antitubulin (Bio-Rad) to monitor protein loading. To detect human A3H, we used an anti-human monoclonal A3H antibody kindly provided by M. Emerman (24).

RESULTS

Mammalian A3Z3 proteins are relatively conserved in comparison to lentiviral Vifs.

All mammals have only one Z3-type A3 domain (Fig. (Fig.1A).1A). We previously described the Z3-type A3 proteins of cattle and sheep and calculated evidence for positive selection in nonhuman lineages (23). Primate and cat A3Z3 proteins also show evidence for positive selection (23, 33, 38). Accordingly, these mammalian A3Z3 proteins show significant variation, ranging from 48% to 88% identity and 62% to 92% similarity (Fig. (Fig.1B).1B). The percent identity and similarity of the mammalian A3Z3 protein sequences correlate with those for evolutionarily related species, where human and rhesus macaque A3Z3 proteins are 83% identical (90% similar) and cow and sheep A3Z3 proteins are 88% identical (92% similar) (Fig. 1A and B). The cat A3Z3 protein is the most removed, ranging from 51% to 52% identity (65% to 66% similarity) with the human and rhesus macaque A3Z3s and 62% identity (74% to 77% similarity) with the cow and sheep A3Z3s. Although A3Z3 proteins (or A3 proteins in general) do not share high sequence identity, as is the case for essential cellular genes like those that encode the core of the ribosome, there are clearly conserved residues and motifs such as the conserved zinc-binding domain found in all A3 proteins and residues unique to A3Z3 proteins (22).

FIG. 1.
Comparison of A3Z3 and Vif sequences. (A) The relatedness of the mammals relevant to this study. The numbers at the branch nodes are approximate divergence times in millions of years. (Adapted from reference 22). (B) Percent sequence identity and similarity ...

In contrast, the Vif proteins of lentiviruses are vastly different in primary amino acid sequence. Alignments of known lentiviral Vif protein sequences show less than 30% identity, even between evolutionarily related viruses (36, 42). However, despite low sequence identity between different Vifs, the proteins share a highly conserved S/TLQY/RLA motif (Fig. (Fig.1C)1C) (36, 42). This virus-specific motif in HIV-1, HIV-2, different primate SIVs, BIV, and MVV Vifs is essential for binding ELOC, which is part of a ubiquitin ligation complex (ELOB/C, CUL2 or CUL5, and RBX) capable of degrading A3s (25). The primate lentiviral Vifs (HIV and SIV) are distinct from the other mammalian Vifs in that they share a zinc-binding domain, HX5CX17-18CX3-5H (HCCH), which binds to CUL5 (25). BIV and MVV Vif lack this domain and therefore do not coimmunoprecipitate with the CUL5 protein when expressed in human cells (25). MVV Vif, on the other hand, coimmunoprecipitates the CUL2 protein, indicating that nonhuman Vifs may utilize a different Cullin (25). Due to broad conservation of the Vif S/TLQY/RLA motif, we constructed triple amino acid substitution derivatives (S/TLQ to AAA) of each lentiviral Vif to use as controls. This mutant is unable to bind ELOC, and it is a common negative control for many HIV-1 and SIV Vif studies (e.g., references 20, 25, 44, and 52).

Representative mammalian A3Z3 proteins can restrict HIV.

Every completely annotated mammalian A3 locus to date has only one copy of a Z3-type A3 gene (A3H-like). We hypothesized that all A3Z3-type proteins are neutralized by their host-specific lentiviral Vif. A3Z3 proteins from human, rhesus macaque, and cat were selected because these mammals are infected with a Vif-containing lentivirus and are capable of restricting HIV or its host lentivirus in the absence of a functional Vif protein (8, 12, 24, 33, 34, 37, 48, 54). The A3Z3 proteins of cow and sheep were also chosen for the representative panel because these artiodactyls are also infected with Vif-containing lentiviruses. Previous studies in our lab have shown that the cow and sheep A3Z2-Z3 can restrict HIV (17). The cow and sheep A3 proteins are also naturally expressed as single domain variants (23), making them more ideal for comparisons with the Z3-type A3s of other mammals.

First, we performed a series of single-cycle HIV-GFP infectivity assays to test whether the single domain cow A3 proteins (A3Z1, A3Z2, and A3Z3) could restrict an HIV-based virus construct. The HIV-GFP viral cocktail was transfected into 293T cells alongside an empty pcDNA3.1 vector or one of the cow A3 proteins in the presence of empty pVR012 vector, BIV Vif-myc, or the BIV VifSLQ-AAA-myc. A portion of the producer cell virus-containing supernatant was used to infect fresh 293T cells to measure infectivity by GFP+ flow cytometry. We were surprised that HIV-GFP was restricted equally well by the cow single domain A3Z3 protein and by the double domain A3Z2-Z3 protein (Fig. (Fig.2A).2A). The cow A3Z1 protein showed no anti-HIV activity, despite being a very active DNA deaminase (23). As controls used previously by many labs, human A3G and mouse A3 both restrict the infectivity of HIV-GFP (e.g., references 17, 21, 27, 50, and 55).

FIG. 2.
Antiretroviral properties of cow A3 proteins and their sensitivity to BIV Vif. (A) Relative infectivity of HIV-GFP produced in the presence of the indicated A3s and a vector control (open bars), BIV Vif (black bars), or BIV VifSLQ-AAA (gray bars). The ...

In parallel experiments, we examined the Vif susceptibility of the cow A3 proteins and found that BIV Vif restores the infectivity of viruses produced in the presence of cow A3Z3 and A3Z2-Z3 but not those produced with human A3G or mouse A3 (Fig. (Fig.2A).2A). These data suggested that BIV Vif might interact only with the cow Z3-type deaminase domain. However, immunoblots of the proteins expressed in the producer cell extracts showed that the Z2-type domain was also clearly susceptible to Vif-mediated degradation despite the fact that this domain is not antiviral (Fig. (Fig.2B2B).

As anticipated by the infectivity data, BIV Vif also degraded the cow A3Z3 and A3Z2-Z3 proteins. None of the cow A3s are degraded in the presence of mutant BIV VifSLQ-AAA-myc (Fig. (Fig.2).2). Overall, these experiments demonstrated that BIV Vif triggers the degradation of both Z2- and Z3-type A3s and that Vif function requires the conserved SLQ motif (and therefore likely also the interaction with ELOC). In addition, the results show that BIV Vif either does not interact with or does not trigger the degradation of the Z1-type A3.

BIV Vif function appears species specific in contrast to that of the related MVV Vif protein.

We next asked whether BIV Vif could neutralize other mammalian A3Z3 proteins besides cow A3Z3. HIV-GFP was produced in the presence of a diverse set of mammalian A3Z3 proteins, and as described above, the producer cells were analyzed by immunoblotting and the resulting viral supernatants were analyzed by infecting susceptible target cells. The A3Z3 proteins showed a range of HIV-GFP restriction phenotypes, with rhesus macaque A3H appearing to have the most potency and the sheep A3Z3 the least (Fig. (Fig.3A).3A). Some of these differences may be due in part to slight variations in protein expression. However, consistent with the species-specific hypothesis, BIV Vif rescued the infectivity only of viruses produced in the presence of cow A3Z3. This counterrestriction effect correlated with greatly reduced cellular cow A3Z3 protein levels. In contrast, even the sheep A3Z3, which is 92% similar to the cow A3Z3, showed no sign of being degraded.

FIG. 3.
Sensitivity of mammalian A3Z3 proteins to BIV and MVV Vif. (A) Relative infectivity of HIV-GFP produced in the presence of the indicated A3s and a vector control (open bars), BIV Vif (black bars), or BIV VifSLQ-AAA (gray bars). The double vector control ...

To ask whether these observations would be reciprocal and possibly generalizable, we turned to the most closely related lentivirus-host interaction, that between MVV Vif and the sheep A3s. The predicted evolutionary distance between the sheep and cow is similar to that between the human and rhesus macaque, approximately 28 million years (Fig. (Fig.1A)1A) (3). BIV and MVV have similar genome organizations (36), and it is reasonable to presume that these viruses arose from a common ancestor (9, 19). However, in comparison to the species-specific activity of BIV Vif, MVV Vif counteracted the restriction activity of nearly all of the A3Z3 proteins (Fig. (Fig.3B).3B). MVV Vif neutralized the sheep and cow A3Z3 proteins with nearly 100% efficiency, and it was also very effective at degrading the human, rhesus macaque, and cat A3Z3 proteins. These effects were equally apparent at the protein level in immunoblots of producer cell extracts. Interestingly, despite MVV Vif having no effect on the restriction capability of human A3G and the mouse A3, protein degradation was also detected for these intended non-single domain A3Z3 controls (human A3G = Z2Z1 and mouse A3 = Z2Z3). These phenotypes are not observed with the MVV VifSLQ-AAA-myc construct, but this may be due to the apparent instability caused by the SLQ mutations (Fig. (Fig.3B).3B). Taken together, these data demonstrate that MVV Vif lacks both species specificity and Z-type specificity, based on its capability of triggering the degradation of a broad array of mammalian A3 proteins that contain different combinations of the Z-type domains.

MVV Vif-mediated degradation of the A3s is not simply an artifact of degrading all cellular proteins, because tubulin levels were unaffected and toxicity was not observed (Fig. (Fig.3B3B and data not shown). Such promiscuity was unanticipated, so we next asked whether MVV Vif has a preference for degrading sheep A3Z3. We analyzed the infectivity of HIV-GFP produced in the presence of sheep A3Z3, human A3H, mouse A3, or human A3G and the range of MVV Vif levels (0, 5, 20, and 50 ng plasmid per transfection). Even the lowest MVV Vif levels were sufficient to rescue HIV-GFP infectivity by counteracting sheep A3Z3 or human A3H (data not shown). However, immunoblot analysis of the producer cell lysates showed that only the sheep A3Z3 was efficiently degraded at the lowest concentrations, suggesting that MVV Vif has some preference for sheep A3Z3.

HIV-1 Vif neutralization of human A3H and other mammalian A3Z3 proteins.

Prior reports have indicated that HIV-1 Vif fails to neutralize rhesus macaque A3H or cat A3Z3 (34, 48). Recent studies with HIV-1 Vif and human A3H (haplotype II) have yielded conflicting results, with some showing neutralization and others not (8, 12, 24, 37, 54). To further investigate the potential interaction between HIV-1 Vif and human A3H and, if apparent, to ask whether it is species specific, we did a series of HIV-GFP infection experiments as described above with HIV-1 Vif from the LAI strain and our panel of mammalian A3Z3s. As observed in a prior study with HIV-1LAI Vif (24), HIV-GFP infectivity was partly restored and the level of cellular human A3H slightly diminished (Fig. (Fig.4A).4A). HIV-GFP infectivity in the presence of the cow, sheep, and mouse A3s was also improved significantly by expressing HIV-1LAI Vif, but this effect was not accompanied by a significant change in cellular protein levels. It is unclear whether all of these effects were dependent on the conserved SLQ motif, because the mutated protein did not express well in comparison to the wild-type protein. Overall, however, the phenotypes attributable to HIV-1LAI Vif were quite modest in comparison to results with other lentiviral Vif proteins or data with HIV-1 Vif and human A3G (Fig. (Fig.22 and and3)3) (see below and also prior studies, e.g., references 6, 29 to 31, 44, 46, 52, and 53).

FIG. 4.
Sensitivity of mammalian A3Z3 proteins to HIV-1LAI Vif. (A) Relative infectivity of HIV-GFP produced in the presence of the indicated A3s and a vector control (open bars), HIV-1LAI Vif (black bars), or HIV-1LAI VifSLQ-AAA (gray bars). The double vector ...

We therefore wondered whether the C-terminal HA epitope tag might be protecting human A3H from Vif-mediated degradation. To address this point, we compared the sensitivities of human A3H-HA and untagged A3H to neutralization by HIV-1LAI Vif (Fig. (Fig.4B).4B). In both instances, the infectivity of HIV-GFP recovered significantly and this effect corresponded to the near disappearance of the A3H-specific band from the cell lysate immunoblot. A similarly large amount of A3H degradation was apparent in analogous experiments with HIV Vif from strain IIIB (data not shown). The only significant difference between the immunoblot experiments shown in Fig. Fig.4A4A and those in Fig. Fig.4B4B is the fact that the former required the use of an anti-HA monoclonal antibody to compare all of the A3s and the latter used an A3H-specific monoclonal antibody to detect the human protein (kindly provided by M. Emerman [24]). To further investigate this suspected difference in antibody sensitivity, the blot in Fig. Fig.4B4B was stripped and probed with the anti-HA antibody and a faint band was detected for the HA-tagged A3H expressed with HIV Vif (data not shown). So the difference in A3 degradation is most likely linked to antibody sensitivity (with the anti-HA monoclonal antibody being more sensitive). Nevertheless, the sum of our results combined to indicate that HIV-1 Vif has a notable preference for human A3H over other Z3-type A3s. The modest neutralization of two of the other A3s may be related to the degradation-independent inhibition noted previously for HIV-1 Vif (18, 39, 40).

SIVmac Vif and FIV Vif differ in their abilities to neutralize other species' A3Z3 proteins.

Many previous studies have investigated the primate A3 neutralization capabilities of SIVmac Vif (5, 26, 27, 41, 48, 51). SIVmac Vif is capable of degrading rhesus macaque A3H, African green monkey A3H, and human A3H (48). To extend these studies, we tested SIVmac Vif against our full panel of mammalian Z3-type A3 proteins (Fig. (Fig.5A).5A). In addition to counteracting the restrictive capabilities of the rhesus macaque and human A3H proteins, SIVmac Vif neutralized cow A3Z3. Again, a corresponding drop in cellular A3 levels was observed. However, SIVmac Vif failed to neutralize the sheep and cat A3Z3 proteins and the mouse A3Z2-Z3 protein. Thus, SIVmac Vif is not quite as promiscuous as the MVV Vif, but it is clearly more so than the BIV or the HIV-1 Vif proteins.

FIG. 5.
Sensitivity of mammalian A3Z3 proteins to SIVmac and FIV Vif. (A) The relative infectivity of HIV-GFP produced in the presence of the indicated A3s and a vector control (open bars), SIVmacVif (black bars), or SIVmac VifSLQ-AAA (gray bars). The double ...

These experiments also revealed another curious phenotype, as the SLQ motif of SIVmac Vif appeared partly dispensable for its counterrestriction effect against the human and rhesus macaque A3H proteins (Fig. (Fig.5A).5A). Together with the aforementioned studies, it is possible that Vif is capable of eliciting two degradation-independent effects—an ELOC-independent mechanism as observed here with SIVmac Vif and an ELOC-dependent mechanism as observed in Fig. Fig.4A4A with HIV-1 Vif. Further studies may be warranted to better understand this potentially degradation-independent mechanism of A3 neutralization.

Finally, to complete the survey, we tested the sensitivity of the mammalian A3Z3 panel to FIV Vif (Fig. (Fig.5B).5B). These results were remarkably parallel to the BIV Vif data, as only the cat A3Z3 protein was neutralized and degraded by FIV Vif. A similar observation was made previously (33). However, our studies uniquely show that FIV Vif-mediated neutralization of the cat A3Z3 protein is dependent on the TLQ motif, indicating a requirement for the ELOC interaction.

DISCUSSION

In contrast to Z1-type A3s, which have not been reported to be degraded by Vif, and Z2-type A3s, which are sometimes degraded by Vif, we have shown here that Z3-type mammalian A3s are invariably degraded by the Vif protein of each species' lentivirus. Vif-mediated degradation of the Z3-type A3s is also accompanied by improved viral infectivity. This strong correlation provides good evidence that the Vif-A3Z3 interaction is relevant to lentiviral replication (i.e., the lentivirus would not have evolved and retained Vif if these mammalian A3Z3s or their ancestral equivalents were not a significant threat). Moreover, our data indicate that the S/TLQ motif of BIV, SIVmac, and FIV Vif is required for degradation of the Z3-type A3s. These data demonstrate that interaction with ELOC is conserved and that the likely mechanism is ubiquitin-mediated degradation. Although the SLQ motif in HIV Vif has been shown to be important for the interaction with ELOC (53), further studies are needed to verify whether this motif is important for HIVLAI Vif and MVV Vif because mutating these residues greatly reduced expression of the mutant forms. The conservation of the Vif-A3Z3 interaction also suggests the existence of at least one common underlying structural determinant, because the lentiviral Vifs are much more divergent at the amino acid level than the A3Z3s of their mammalian host (Fig. (Fig.11).

A second major conclusion to stem from our studies is that some lentiviral Vif proteins lack species specificity (summarized in Table Table11 ). The best example is MVV Vif, which triggers the near-complete degradation of the sheep, cow, and cat A3Z3 proteins and partial degradation of the human and rhesus macaque A3H proteins. MVV Vif also restores or improves HIV infectivity in the presence of each mammalian A3Z3 protein. In contrast, the closest relative to this protein, BIV Vif, appears to neutralize only the cow A3Z3 protein. A second example is SIVmac Vif, which triggers the degradation of the rhesus macaque, human, and cow A3Z3 proteins and provides a corresponding improvement in virus infectivity. Again, in contrast, the closest relative to this protein, HIV-1 Vif, appears fairly specific to the human A3H protein. It is probable that had we expanded the survey to include an even broader range of mammalian A3s, each lentiviral Vif would be able to trigger the degradation of multiple A3s (i.e., the Vif-A3Z3 interaction appears to be far from species specific). It is further probable that Vifs from different lentiviral strains within a species will possess a range of A3 neutralization capabilities, as has been indicated by the different strains of HIV-1 (28).

TABLE 1.
Qualitative summary of Vif-mediated A3 degradation and rescue of HIV-GFP infectivity from A3 restriction

Another apparent trend in our data set is that Vif may have the capacity to antagonize the restriction activity of a given A3Z3 either without triggering its degradation or by utilizing a different degradation pathway. First, we observed an increase in HIV-GFP infectivity when HIV-1LAI Vif was coexpressed with cow A3Z3, sheep A3Z3, or mouse A3Z2-Z3, even in the absence of detectable protein degradation. It may be possible that HIV-1 Vif is interacting in a way that hinders the A3 protein's ability to restrict but is not efficiently recruiting or correctly positioning the cellular degradation machinery complex (18, 39, 40). Second, we observed that the SIVmac VifSLQ-AAA mutant is capable of triggering partial degradation of the human and rhesus macaque A3H proteins. It is therefore possible that Vif may bind to A3Z3 proteins and recruit an alternative cellular complex to induce degradation. Other possibilities are that degradation may depend in part on species-specific interactions or that the Vif variants analyzed here may not be fully representative of the many other variants that exist naturally.

The A3Z3-type deaminase domain can be expressed on its own and show antiviral activity. It can also be expressed as part of a double-domain deaminase (Z2-Z3) in many mammals, including cattle, sheep, pigs, cats, and mice. The cow, sheep, and cat A3Z2-Z3 proteins all restrict HIV and/or FIV and are neutralized by its host-specific Vif (17, 33, 34). We show in this study that the cow A3Z2 protein is degraded in the presence of BIV Vif, even though it does not restrict HIV (Fig. (Fig.2).2). It is therefore possible that Vif is able to simultaneously interact with both types of Z domains (the A3Z2-Z3 protein), resulting in a synergistic neutralization effect. Additional studies will be needed to systematically address this possibility and test alternatives such as additive neutralization or competitive binding.

Finally, our data clearly demonstrate that the Vif-A3Z3 interaction is not species specific but more likely to be host optimized and considerably promiscuous. The idea of a species-specific A3-Vif interaction arose following seminal work that mapped a key residue in A3G, D128, which appeared to dictate the susceptibility of this protein to Vif-mediated degradation (4, 26, 27, 41, 51). Here, our broad survey suggests that lentiviral Vifs are optimized to interact with their host species' A3Z3 proteins because in all instances the lentiviral Vifs can effectively degrade and neutralize the A3Z3 of their host. However, we also observe that this interaction can be highly versatile and, in multiple instances, Vif is able to recognize and degrade A3 proteins of other species, some from which it is far removed (at least in evolutionary terms [Fig. [Fig.1A]).1A]). Therefore, while Vif invariably functions as an A3 antagonist in its host species, its ability to neutralize A3s of other species provides a mechanism to overcome this key barrier to zoonotic transmission. Additional comparative studies are likely to help map critical interacting regions and inform efforts to develop novel antiviral drugs that leverage the Vif-A3 axis.

Acknowledgments

We thank J. Albin, J. Hultquist, and V. Pathak for comments on the manuscript; M. Emerman for key reagents; X-F. Yu for sharing unpublished data; and J. Weaver for providing technical assistance.

R. LaRue is a student in the Comparative Molecular Bioscience program and is supported in part by a studentship from the MN Agricultural Experiment Station (MAES) and College of Veterinary Medicine (CVM). This work was funded by a grant from the National Institutes of Health AI064046.

We dedicate this paper to David Derse, who enthusiastically supported our nonhuman A3 studies over the years.

Footnotes

[down-pointing small open triangle]Published ahead of print on 2 June 2010.

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