Identification of a Novel WxSLVK Motif in the N Terminus of Human Immunodeficiency Virus and Simian Immunodeficiency Virus Vif That Is Critical for APOBEC3G and APOBEC3F Neutralization

The function of lentiviral Vif proteins is to neutralize the host antiviral cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F). Vif bridges a cullin 5-based E3 ubiquitin ligase with A3G and A3F and mediates their degradation by proteasomes. Recent studies have found that Vif uses different domains to bind to A3G and A3F. A ¹⁴DRMR¹⁷ domain binds to A3F, ⁴⁰YRHHY⁴⁴ binds to A3G, and ⁶⁹YxxL⁷² binds to both A3G and A3F. Here, we report another functional domain of Vif. Previously, we demonstrated that human immunodeficiency virus type 1 (HIV-1) Vif failed to mediate A3G proteasomal degradation when all 16 lysines were mutated to arginines. Here, we show that K26, and to a lesser extent K22, is critical for A3G neutralization. K22 and K26 are part of a conserved ²¹WxSLVK²⁶ (x represents N, K, or H) motif that is found in most primate lentiviruses and that shows species-specific variation. Both K22 and K26 in this motif regulated Vif specificity only for A3G, whereas the SLV residues regulated Vif specificity for both A3F and A3G. Interestingly, SLV and K26 in HIV-1 Vif did not directly mediate Vif interaction with either A3G or A3F. Previously, other groups have reported an important role for W21 in A3F and A3G neutralization. Thus, ²¹WxSLVK²⁶ is a novel functional domain that regulates Vif activity toward both A3F and A3G and is a potential drug target to inhibit Vif activity and block HIV-1 replication.The replication of human immunodeficiency virus type 1 (HIV-1) is seriously impaired in human primary lymphocytes when the viral protein Vif is not present (8, 38). The first cellular target of Vif was identified as APOBEC3G (A3G) (34), which belongs to the cytidine deaminase family known as APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide) (14). This family consists of APOBEC1; activation-induced deaminase (AID); APOBEC2; a subgroup of APOBEC3 (A3) proteins, including A3A, A3B, A3C, A3DE, A3F, A3G, and A3H; and APOBEC4 in humans (12). They have one or two copies of a cytidine deaminase domain with a signature motif (HxEx_23-28PCx_2-4C), and normally only one of the cytidine deaminase domains has deaminase activity.All seven A3 genes have been shown to inhibit the replication of various types of retroviruses via cytidine deamination-dependent or -independent mechanisms (3). In particular, A3B, A3DE, A3F, and A3G inhibit HIV-1 replication, whereas A3A and A3C do not (1, 6, 7, 19, 34, 42, 50). Recently, it was shown that optimizing A3H expression in cell culture also inhibits HIV-1 replication (4, 10, 25, 39). Among these proteins, A3G and A3F have the most potent anti-HIV-1 activities. A3G and A3F share ∼50% sequence similarity but have different biochemical properties (41) and different target sequence preferences while catalyzing cytidine deamination of viral cDNAs (19).Nevertheless, HIV-1 is able to elude this defense mechanism and cause human disease for two reasons. First, A3B and A3H are expressed only at low levels in vivo (4, 7, 18, 26). Second, HIV-1 produces Vif, which is expressed in all lentiviruses except equine infectious anemia virus. Vif can destabilize A3DE, A3F, and A3G proteins by targeting them to the proteasomal degradation pathway (6, 22, 35, 37, 50). In addition, Vif may also inhibit A3 activity independently of proteasomal degradation (15, 16, 31).The action of Vif is highly species specific. Vif from HIV-1 inactivates only A3G from humans, and Vif from simian immunodeficiency virus (SIV) isolated from African green monkeys (AGM) does not inactivate A3G from humans. Nevertheless, Vif from SIV isolated from rhesus macaques (MAC) inactivates A3G from all humans, AGM, and MAC (21). A single residue in A3G at position 128, an aspartic acid in humans versus a lysine in AGM, determines A3G sensitivity to HIV-1 Vif (2, 32, 44). In addition, an N-terminal domain in HIV-1 Vif, ¹⁴DRMR¹⁷, determines Vif specificity for different A3G proteins (33).Vif targets A3G to the proteasome by acting as an adaptor protein that bridges A3G with a cullin 5 (Cul5)-based E3 ubiquitin ligase complex, which includes Cul5, elongin B (EloB), and EloC (46). Vif has a BC box motif (¹⁴⁴S¹⁴⁵L¹⁴⁶Q) that binds to EloC (23, 47) and an HCCH motif (¹¹⁴C/¹³³C) that binds to Cul5 (20, 24, 43). It has also been shown that Vif specifically binds to a region from amino acids 126 to 132 of A3G and to amino acids 283 to 300 of A3F (13, 30). It is believed that as a consequence of these interactions, A3G is polyubiquitylated and directed to 26S proteasomes for degradation.Several domains that determine Vif interactions with A3F and A3G have been identified. Analysis of HIV-1 patient-derived Vif sequences initially found that W11 is essential for A3F recognition and K22, Y40, and E45 are required for A3G recognition (36). The previously identified agmA3G-specific ¹⁴DRMR¹⁷ domain was also found to determine Vif specificity for A3F (33) by direct binding (29). An A3G-specific binding domain, ⁴⁰YRHHY⁴⁴, has also been identified (29), and a ⁶⁹YxxL⁷² domain interacts with both A3G and A3F (11, 28, 45).We have previously shown that Vif can mediate A3G proteasomal degradation in the absence of A3G polyubiquitylation and that, unexpectedly, this process is dependent on lysines in Vif (5). Here, we identify two N-terminal lysines that are important for Vif function. We show that these lysines are part of a ²¹WxSLVK²⁶ motif that is conserved in Vif from primate lentiviruses and that this motif regulates Vif activities against both A3G and A3F via different mechanisms.     

Identification of a Critical T(Q/D/E)x5ADx2(I/L) Motif from Primate Lentivirus Vif Proteins That Regulate APOBEC3G and APOBEC3F Neutralizing Activity

Primate lentiviruses are unique in that they produce several accessory proteins to help in the establishment of productive viral infection. The major function of these proteins is to clear host resistance factors that inhibit viral replication. Vif is one of these proteins. It functions as an adaptor that binds to the cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) and bridges them to a cullin 5 (Cul5) and elongin (Elo) B/C E3 ubiquitin ligase complex for proteasomal degradation. So far, 11 discontinuous domains in Vif have been identified that regulate this degradation process. Here we report another domain, T(Q/D/E)x₅ADx₂(I/L), which is located at residues 96 to 107 in the human immunodeficiency virus type 1 (HIV-1) Vif protein. This domain is conserved not only in all HIV-1 subtypes but also in other primate lentiviruses, including HIV-2 and simian immunodeficiency virus (SIV), which infects rhesus macaques (SIVmac) and African green monkeys (SIVagm). Mutations of the critical residues in this motif seriously disrupted Vif''s neutralizing activity toward both A3G and A3F. This motif regulates Vif interaction not only with A3G and A3F but also with Cul5. When this motif was inactivated in the HIV-1 genome, Vif failed to exclude A3G and A3F from virions, resulting in abortive HIV replication in nonpermissive human T cells. Thus, T(Q/D/E)x₅ADx₂(I/L) is a critical functional motif that directly supports the adaptor function of Vif and is an attractive target for inhibition of Vif function.Vif is a small viral protein that has 192 amino acids and is expressed by most lentiviruses, except for equine infectious anemia virus. It was first discovered in human immunodeficiency virus type 1 (HIV-1) (13, 14, 31), and its function in HIV-1 infection has been studied extensively (9, 34). Infection of human T-cell lines with vif-defective (ΔVif) HIV-1 identified two different cell types, namely, permissive cells that can be infected by ΔVif HIV-1 and nonpermissive cells, which are resistant to ΔVif HIV-1 (10, 36). Genomic complementation analysis indicated that these nonpermissive cells express a Vif-sensitive dominant viral inhibitor(s) (17, 27). The first inhibitor identified was APOBEC3G (A3G) (25), which belongs to the APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide) family. In humans, this family consists of APOBEC1; activation-induced deaminase (AID); APOBEC2; a subgroup of APOBEC3 (A3) proteins, including A3A, A3B, A3C, A3DE, A3F, A3G, and A3H; and APOBEC4. All seven A3 genes have been shown to inhibit replication of various types of retrovirus by cytidine deamination-dependent and -independent mechanisms, as reviewed recently (21, 30, 35). In particular, human A3B, A3DE, A3F, A3G, and A3H inhibit HIV-1 replication, whereas A3A and A3C do not (2, 5, 6, 25, 39, 46). Among these, the protein expression of A3G and A3F in human primary tissues has been demonstrated, and in vitro studies indicate that these proteins have the most potent anti-HIV-1 activities. A3G and A3F share ∼50% sequence similarity but have different biochemical properties (38) and different target sequence preferences while catalyzing cytidine deamination of viral cDNAs (15).Vif hijacks the cellular proteasomal machinery to destroy A3G and A3F by the protein degradation pathway (18, 26, 33, 46). Vif acts as an adaptor protein that bridges A3 proteins to a cullin 5 (Cul5)-based E3 ubiquitin ligase complex, which includes Cul5, EloB, and EloC (44). These interactions trigger the polyubiquitylation of Vif, A3G, and A3F and direct them to 26S proteasomes for degradation. Thus, Vif binding to A3G or A3F as well as to Cul5/EloBC is a critical step for A3G and A3F degradation. Although A3G and A3F share a high level of homology, different surfaces are used for Vif interaction. Vif binds to the N-terminal region of A3G, from residues 126 to 132, and to the C-terminal region of A3F, from residues 283 to 300 (12, 24). In addition, 11 discontinuous motifs in the Vif protein have been identified as regulating Vif interactions with A3G, A3F, or the Cul5/EloBC E3 ligase complex. Three motifs determine Vif interaction with the E3 ligase. The ¹⁰⁸Hx₅Cx_17-18Cx_3-5H¹³⁹ motif, also called the HCCH zinc finger, binds to Cul5 (16, 20, 41); the ¹⁴⁴SLQYLA¹⁴⁹ motif, which is also called the BC box, binds to EloC (19, 45); and the ¹⁶¹PPLPx₄L¹⁶⁹ motif, which is also called the Cul box, binds to Cul5 (32, 45). The ¹⁶¹PPLP¹⁶⁴ subdomain has multiple activities, which not only determine Vif dimerization (43) but also regulate Vif binding to A3G (8, 37) and EloB (1). The other 8 motifs regulate the interaction between Vif and A3G/A3F. The ²¹WxSLVK²⁶ (3, 7) and ⁴⁰YRHHY⁴⁴ (23) motifs regulate Vif binding to A3G; the ¹¹Wx₂DRMR¹⁷ (23), ⁷⁴TGERxW⁷⁹ (11), and ¹⁷¹EDRW¹⁷⁴ (4) motifs regulate Vif binding to A3F; and the ⁵⁵VxIPLx₄L⁶⁴ (11), ⁶⁹YxxL⁷² (22), and ⁸¹LGxGx₂IxW⁸⁹ (4) motifs regulate Vif binding to both A3G and A3F. The ⁸¹LGxGx₂IxW⁸⁹ motif also regulates Vif binding to Cul5 (4). Thus, HIV has developed rather complicated mechanisms to assemble a protein degradation complex to neutralize these two critical host factors. A full understanding of these mechanisms is essential for pharmaceutical inhibition of Vif function to prevent HIV-1 infection. Here we report another functional motif from a previously uncharacterized region of HIV-1 Vif that regulates Vif interactions with A3G, A3F, and the Cul5/EloBC E3 ligase complex. Since this Vif region is the only one left uncharacterized, this is a significant step toward a complete understanding of this important host-pathogen interaction.     

Remarkable Lethal G-to-A Mutations in vif-Proficient HIV-1 Provirus by Individual APOBEC3 Proteins in Humanized Mice

Genomic hypermutation of RNA viruses, including human immunodeficiency virus type 1 (HIV-1), can be provoked by intrinsic and extrinsic pressures, which lead to the inhibition of viral replication and/or the progression of viral diversity. Human APOBEC3G was identified as an HIV-1 restriction factor, which edits nascent HIV-1 DNA by inducing G-to-A hypermutations and debilitates the infectivity of vif-deficient HIV-1. On the other hand, HIV-1 Vif protein has the robust potential to degrade APOBEC3G protein. Although subsequent investigations have revealed that lines of APOBEC3 family proteins have the capacity to mutate HIV-1 DNA, it remains unclear whether these endogenous APOBEC3s, including APOBEC3G, contribute to mutations of vif-proficient HIV-1 provirus in vivo and, if so, what is the significance of these mutations. In this study, we use a human hematopoietic stem cell-transplanted humanized mouse (NOG-hCD34 mouse) model and demonstrate the predominant accumulation of G-to-A mutations in vif-proficient HIV-1 provirus displaying characteristics of APOBEC3-mediated mutagenesis. Notably, the APOBEC3-associated G-to-A mutation of HIV-1 DNA that leads to the termination of translation was significantly observed. We further provide a novel insight suggesting that HIV-1 G-to-A hypermutation is independently induced by individual APOBEC3 proteins. In contrast to the prominent mutation in intracellular proviral DNA, viral RNA in plasma possessed fewer G-to-A mutations. Taken together, these results provide the evidence indicating that endogenous APOBEC3s are associated with G-to-A mutation of HIV-1 provirus in vivo, which can result in the abrogation of HIV-1 infection.Human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3 [A3]) family proteins are potent mutators of a broad spectrum of retroviruses, including human immunodeficiency virus type 1 (HIV-1) (4, 5, 13, 16, 29, 61). A3s are cellular cytidine deaminases that convert C in the viral minus-strand cDNA to U, resulting in the alteration of G to A in the nascent proviral DNA. Several A3 proteins are incorporated into progeny virions and mutate viral cDNA in the invaded cells, which is thought to result in the inhibition of viral replication (4, 5, 13, 16, 29, 46, 61). On the other hand, an HIV-1 accessory protein, viral infectivity factor (Vif), has the ability to counteract the incorporation of certain A3 proteins such as A3G and A3F into progeny virions by degrading these proteins through the proteasome-dependent pathway (31, 45, 47, 50). Lines of in vitro investigations have elucidated the mechanisms of G-to-A hypermutation of HIV-1 DNA mediated by A3s and the counteracting ability of Vif against A3s, which have shed light on the relevance of host-retrovirus interaction (4, 5, 21, 59, 60). Nevertheless, the physiological balance between intrinsic A3s and Vif in vivo is poorly understood, and the significance of A3-mediated mutagenesis for HIV-1 replication in vivo remains unresolved.In order to investigate the dynamics of human-specific pathogens in vivo, we have recently constructed a humanized mouse (NOG-hCD34 mouse) model by xenotransplanting human CD34⁺ hematopoietic stem cells into an immunodeficient NOD/SCID/IL-2R-γ^null (NOG) mouse (15, 34). In the humanized mice, human leukocytes, including human CD4⁺ T cells, are successfully differentiated de novo and are stably and longitudinally maintained for more than 1 year (15, 34). By utilizing the humanized mice, we have established a novel animal model for HIV-1 infection (34). Our humanized mice are capable of supporting persistent replication of CCR5-tropic HIV-1 for more than 7 months and mirror the characteristics of HIV-1 pathogenesis, such as the depletion of memory CD4⁺ T cells in the periphery and the preferential infection of effector memory T cells (34).Recently, Ince et al. reported the significance of HIV-1 mutation and its influence on HIV-1 expansion by using a humanized mouse model system (14). In that paper, however, the authors particularly focused on the diversity of the HIV-1 env gene, and therefore, the involvement and the significance of A3-associated mutagenesis in HIV-1 expansion in vivo remain unclear.In this study, by using the humanized mouse (NOG-hCD34 mouse) model, we show that G-to-A mutation of vif-proficient HIV-1 provirus exhibiting the characteristics of A3-mediated mutagenesis occurs in vivo. We also provide a novel insight indicating that intrinsic A3-mediated G-to-A mutation is independently caused by endogenous A3 protein. Furthermore, in contrast to the prominent accumulation of G-to-A mutation in provirus, we observed few mutations in virion-associated RNA in plasma. Based on our findings, we discuss the possibility that endogenous A3s have a significant influence on HIV-1 infection in vivo.     

Lentiviral Vif Degrades the APOBEC3Z3/APOBEC3H Protein of Its Mammalian Host and Is Capable of Cross-Species Activity

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, SIV_mac 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 SIV_agm 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 SIV_agm 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 SIV_mac or HIV-2 (41, 51). Second, SIV_mac 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-x_23-28-C-x_2-4-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 SIV_agm and is susceptible to degradation by SIV_agm 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 SIV_mac 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.     

Identification of 81LGxGxxIxW89 and 171EDRW174 Domains from Human Immunodeficiency Virus Type 1 Vif That Regulate APOBEC3G and APOBEC3F Neutralizing Activity

The human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) potently restrict human immunodeficiency virus type 1 (HIV-1) replication, but they are neutralized by the viral protein Vif. Vif bridges A3G and A3F with a Cullin 5 (Cul5)-based E3 ubiquitin ligase and mediates their proteasomal degradation. This mechanism has been extensively studied, and several Vif domains have been identified that are critical for A3G and A3F neutralization. Here, we identified two additional domains. Via sequence analysis of more than 2,000 different HIV-1 Vif proteins, we identified two highly conserved amino acid sequences, ⁸¹LGxGxSIEW⁸⁹ and ¹⁷¹EDRWN¹⁷⁵. Within the ⁸¹LGxGxSIEW⁸⁹ sequence, residues L81, G82, G84, and, to a lesser extent, I87 and W89 play very critical roles in A3G/A3F neutralization. In particular, residues L81 and G82 determine Vif binding to A3F, residue G84 determines Vif binding to both A3G and A3F, and residues ⁸⁶SIEW⁸⁹ affect Vif binding to A3F, A3G, and Cul5. Accordingly, this ⁸¹LGxGxSIEW⁸⁹ sequence was designated the ⁸¹LGxGxxIxW⁸⁹ domain. Within the ¹⁷¹EDRWN¹⁷⁵ sequence, all residues except N175 are almost equally important for regulation of A3F neutralization, and consistently, they determine Vif binding only to A3F. Accordingly, this domain was designated ¹⁷¹EDRW¹⁷⁴. The LGxGxxIxW domain is also partially conserved in simian immunodeficiency virus Vif from rhesus macaques (SIVmac239) and has a similar activity. Thus, ⁸¹LGxGxxIxW⁸⁹ and ¹⁷¹EDRW¹⁷⁴ are two novel functional domains that are very critical for Vif function. They could become new targets for inhibition of Vif activity during HIV replication.The function of the lentiviral protein Vif is to neutralize the major host antiretroviral cytidine deaminases that belong to the APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide) family, as recently reviewed by several investigators (18, 29, 31). This family consists of APOBEC1; activation-induced deaminase (AID); APOBEC2; a subgroup of APOBEC3 (A3) proteins, including A3A, A3B, A3C, A3DE, A3F, A3G, and A3H; and APOBEC4 in humans. They have one or two copies of a cytidine deaminase (CDA) domain with a signature motif (HxEx_23-28PCx_2-4C), only one of which normally has deaminase activity.All seven A3 genes have been shown to inhibit replication of various types of retrovirus via cytidine deamination-dependent or -independent mechanisms. In particular, human A3B, A3DE, A3F, A3G, and A3H inhibit human immunodeficiency virus type 1 (HIV-1) replication, whereas A3A and A3C do not (1, 3, 5, 26, 33, 37). Among these proteins, the expression of human A3G and A3F in vivo has been demonstrated, and in vitro studies indicate that they have the most potent anti-HIV-1 activity. A3G and A3F share ∼50% sequence similarity but have different biochemical properties (32) and different target sequence preferences while catalyzing cytidine deamination of viral cDNAs (13). Expression of human A3B has not been detected (7), and a 29.5-kb deletion spanning from the 3′ end of the A3A gene to the 8th exon of the A3B gene, leading to the complete removal of the A3B gene, has been detected in certain human populations (12). Human A3H is also poorly expressed in vivo (20). It was reported that human A3H has four haplotypes (Hap I, II, III, and IV), and only Hap II, which is maintained primarily in African populations, could be stably expressed in vitro (19). However, expression of this protein has not been detected in any human populations. Thus, the primary function of HIV-1 Vif is to neutralize A3G, A3F, and, to a lesser extent, A3DE.Vif hijacks cellular proteasomal machinery to destroy these host cytidine deaminases by protein degradation (15, 27, 30). Vif acts as an adaptor protein that bridges A3 proteins with a Cullin 5-based E3 ubiquitin ligase complex, which includes Cul5, Elongin B (EloB), and Elongin C (EloC) (35). Vif has a BC-box motif (¹⁴⁴SLQYLALA¹⁴⁹) that binds to EloC (16, 36) and an HCCH motif (¹⁰⁸Hx₅Cx_17-18Cx_3-5H¹³⁹) that binds to Cul5 (14, 17, 34). On the other hand, Vif also interacts with A3G and A3F. As a consequence of these interactions, A3G and A3F are polyubiquitylated and directed to 26S proteasomes for degradation. In addition, Vif may also inhibit A3 activity independently of proteasomal degradation (10, 11, 24).Interactions between Vif and A3G/A3F are a key step for their proteasomal degradation, and this mechanism has been extensively studied. First, unique surfaces in A3G and A3F important for Vif interaction were identified, and interestingly, they are located in different regions of the two proteins (9, 23). Second, several discontinuous surfaces on Vif have been found to regulate A3G and/or A3F degradation. The ⁴⁰YRHHY⁴⁴ domain specifically binds to A3G and determines Vif specificity for A3G (22); the ¹¹WxxDRMR¹⁷ and ⁷⁴TGERxW⁷⁹ domains specifically bind to A3F and determine Vif specificity for A3F (8, 22); and the ²¹WxSLVK²⁶, ⁵⁵VxIPLx₄L⁶⁴, and ⁶⁹YxxL⁷²domains determine Vif specificity for both A3G and A3F (2, 6, 8, 21). These results indicate that the mechanism that regulates Vif recognition of A3G and A3F is quite complicated, and understanding this mechanism is critical for pharmaceutical protection of A3G and A3F from Vif-mediated proteasomal degradation.Based on our current knowledge of these functional domains, it has been thought that Vif interacts with A3G and A3F mainly via its N-terminal region and with Cul5 E3 ubiquitin ligase machinery via its C-terminal region. However, here we identify a new A3G and A3F regulatory domain from the central region and a new A3F regulatory domain from the C-terminal region of HIV-1 Vif. Our results indicate that A3G and A3F interaction surfaces on HIV-1 Vif are structurally complex, and more efforts are required for a complete understanding of this host-pathogen interactive mechanism.     

The Glycosylated Gag Protein of a Murine Leukemia Virus Inhibits the Antiretroviral Function of APOBEC3

APOBEC proteins have evolved as innate defenses against retroviral infections. Human immunodeficiency virus (HIV) encodes the Vif protein to evade human APOBEC3G; however, mouse retroviruses do not encode a Vif homologue, and it has not been understood how they evade mouse APOBEC3. We report here a murine leukemia virus (MuLV) that utilizes its glycosylated Gag protein (gGag) to evade APOBEC3. gGag is critical for infection of in vitro cell lines in the presence of APOBEC3. Furthermore, a gGag-deficient virus restricted for replication in wild-type mice replicates efficiently in APOBEC3 knockout mice, implying a novel role of gGag in circumventing the action of APOBEC3 in vivo.APOBEC3G (hA3G) in humans and its mouse orthologue, APOBEC3 (mA3), act as potent innate defenses against retroviral infection. Both proteins deaminate cytidine in single-stranded DNA, ultimately resulting in hypermutation of newly synthesized proviral DNA (6, 16), although additional deaminase-independent mechanisms of inhibition have been identified (2). Infectious exogenous retroviruses, including human immunodeficiency virus (HIV) and murine leukemia viruses (MuLVs), have evolved mechanisms to circumvent the action of the APOBEC proteins (3, 6). HIV encodes the Vif protein, which facilitates the rapid proteolysis of hA3G, while the mechanism by which exogenous MuLVs evade the action of mA3 is unknown (6).Exogenous MuLVs, as well as some other gammaretroviruses, encode a glycosylated Gag protein (gGag) originating from an alternate translation start site upstream of the methionine start site of the Gag structural polyproteins (10, 17, 27). gGag is synthesized at similar rates and levels as the structural Gag polyprotein in MuLV-infected cells but is glycosylated and undergoes distinct proteolytic processing (10, 12, 21). A carboxyl fragment of gGag is released from the cell, while an amino fragment is incorporated into the plasma membrane as a type 2 transmembrane protein (12, 25). The functions of gGag remain unclear, but mutations that eliminate its synthesis severely impede in vivo replication of the virus with little, if any, effect on replication in fibroblastic cell lines (7, 19, 26). APOBEC3 proteins are expressed in many tissues in vivo but are poorly expressed in many in vitro cell lines (6), suggesting a possible link between gGag expression and the evasion of mA3 by MuLVs. These studies were undertaken to determine if the expression of the gGag protein facilitated MuLV replication in the presence of mA3 in vitro and in vivo.     

Dissection of the HIV Vif Interaction with Human E3 Ubiquitin Ligase

The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly in vitro using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.HIV Vif antagonizes the human antiviral protein APOBEC3G by hijacking the human Elongin B/C (EloBC)-cullin-SOCS box (ECS)-type E3 ubiquitin ligase, resulting in the polyubiquitination of APOBEC3G and subsequently its proteasomal degradation. Canonical ECS-type ubiquitin ligases consist of a cullin scaffold protein to which adaptor and substrate receptor proteins bind at the N terminus. HIV Vif serves as a substrate receptor protein—its N terminus recruits APOBEC3G, while multiple C-terminal regions assemble with the E3 ligase (9, 13, 24). The E3 ligase interacting regions include a zinc finger (residues 100 to 140), a BC box (residues 141 to 154), and a cullin box (residues 155 to 176) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.(A) A sequence schematic of Vif showing the regions that interact with A3G, A3F, EloBC, and Cul5. (B) An illustration of the assembly of the Vif-E3 ubiquitin ligase. (C) A homology model of Vif-Cul5-EloBC, where the Vif BC box-EloBC is actual structural data (PDB ID 3DCG).Vif binds the cullin adaptor proteins EloB and EloC through the BC-box region (24). The BC box is a loop-helix motif with the consensus sequence (T/S)LxxxCxxx(V/L/I) (7), and it also exists in cellular proteins that interact with EloBC. While Vif does not fit this consensus perfectly, it still binds EloBC with high affinity, and this interaction is lost upon mutation or deletion of consensus BC-box residues (10, 24, 25). This interaction has been described previously for the cellular proteins VHL (15), SOCS2 (3), SOCS3 (1), SOCS4 (4), and recently HIV Vif (14).Both the Vif zinc finger and cullin box interact with the E3 ligase scaffold protein cullin 5 (Cul5) (11, 12, 20, 21). It has been established that the zinc finger is required for Vif to bind Cul5. Mutation of critical histidine or cysteine residues in this region or the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) abolishes the Vif-Cul5 interaction (8, 11, 25). The sequence of the Vif cullin box is not as conserved as those of cellular SOCS-box proteins, which have a defined structure and determine the specificities of their respective cullins (6). The role of the Vif cullin box is not clear, but it has been suggested to promote dimerization of Vif, involving the conserved PPLP region (22, 23), and has recently been implicated in APOBEC3G binding (5, 17). While its importance in Cul5 binding has been demonstrated in coimmunoprecipitation experiments (14), experimental data also exist showing that the Vif zinc finger alone still immunoprecipitates Cul5 (11, 21).To dissect the assembly of the Vif-E3 ubiquitin ligase, we quantified the binding interactions between various C-terminal Vif constructs, EloBC, and Cul5 by isothermal titration calorimetry (ITC) and fluorescence polarization (FP). We additionally probed the effects of the cullin box on Vif dimerization.     

The Transmembrane Domain of BST-2 Determines Its Sensitivity to Down-Modulation by Human Immunodeficiency Virus Type 1 Vpu

Bone marrow stromal cell antigen 2 (BST-2, also known as tetherin) restricts the production of a number of enveloped viruses by blocking virus release from the cell surface. This antiviral activity is counteracted by such viral factors as Vpu of human immunodeficiency virus type 1 (HIV-1). Here, we report that Vpu antagonizes human BST-2 but not BST-2 derived from African green monkeys. The determinants of susceptibility to Vpu map to the transmembrane domain of BST-2. In accordance with this, expression of human BST-2 containing a modified transmembrane domain effectively blocks the replication of wild-type Vpu-expressing HIV-1 in CD4⁺ T cells. Furthermore, these BST-2 variants, as opposed to wild-type human BST-2, are refractory to Vpu-mediated down-regulation as a result of an attenuated interaction with Vpu. In view of the work by others pointing to a key role of the transmembrane domain of Vpu in promoting virus release, our data suggest that a direct interaction through the transmembrane domain of each of these two proteins is a prerequisite for Vpu to down-modulate BST-2.Human immunodeficiency virus type 1 (HIV-1) encodes four accessory proteins, Vif, Vpr, Vpu, and Nef. Although they are dispensable for HIV-1 replication in certain transformed cell lines, these accessory proteins play important roles in HIV-1 pathogenesis by modulating host immunity and overcoming antagonism by cellular factors (10). For example, Vif counteracts APOBEC3G by recruiting the cullin 5-elongin B/C ubiquitin ligase complex and sending polyubiquitinated APOBEC3G to proteasomes for degradation (29). In the absence of Vif, newly synthesized APOBEC3G is incorporated into virus particles and hampers the production of infectious proviral DNA in the new round of infection (4, 10, 23). In addition to its role in down-modulating the cell surface expression of CD4 in infected T cells (11), Vpu stimulates HIV-1 production in cells such as HeLa cells (26). The mechanism behind this latter activity of Vpu was unknown until it was recently discovered that bone marrow stromal cell antigen 2 (BST-2, also known as tetherin, CD317, or HM1.24) blocks the release of HIV-1 and that this inhibitory effect is antagonized by viral Vpu (16, 25).BST-2 harbors an N-terminal transmembrane domain and a C-terminal glycosyl-phosphatidylinositol anchor that together create an unusual topology with both termini of BST-2 inserted into the plasma membrane (8, 18). This unique topology of BST-2 may underlie the mechanism for the retention of progeny virus particles at the cell surface (16). An indirect mechanism behind this tethering effect has not been ruled out, especially in view of the difficulty of detecting BST-2 protein in purified HIV-1 particles (14). In addition to HIV-1, a number of enveloped viruses are subject to inhibition by BST-2, including simian immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus, Mason-Pfizer monkey virus, and Lassa virus, as well as Ebola and Marburg viruses (5, 6, 16, 19, 25). This suggests that BST-2 has a broad antiviral effect spectrum.The bst-2 gene has in its promoter the IRF-1/2 and ISGF3 response elements and thus belongs to the interferon-stimulated gene family (17). In line with its ability to impair the release of enveloped viruses, BST-2 has been demonstrated to be the effector in human embryonic kidney (HEK293T) cells that leads to the interferon-induced block of Vpu deletion-containing HIV-1 production (15). However, the African green monkey kidney cell line COS-7 responds to interferon treatment with a different outcome in that the production of both Vpu deletion-containing and Vpu-expressing HIV-1 is inhibited (15). This indicates that interferon induces a block to HIV-1 in COS-7 cells that cannot be overcome by Vpu. A conceivable candidate that creates this block is BST-2 in COS-7 cells (hereafter named agmBST-2). In this study, we provide evidence that depletion of endogenous BST-2 in COS-7 cells greatly alleviates interferon-induced inhibition of HIV-1 production. The refractoriness of agmBST-2 to Vpu results from a weak association of these two proteins and a resistance of agmBST-2 to Vpu-mediated down-regulation.