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.     

Acutely Transforming Retrovirus Expressing Nras Generated from HT-1080 Fibrosarcoma Cells Infected with the Human Retrovirus XMRV

Virus from HT-1080 fibrosarcoma cells infected with the human retrovirus XMRV (xenotropic murine leukemia virus-related virus) can induce rare foci of transformation in rat 208F fibroblasts. Characterization of three such foci revealed that one produced an acutely transforming virus at a high titer. The virus consists of a mutant Nras cDNA from the HT-1080 cells inserted into a retroviral vector (added to the HT-1080 cells as a marker for infection) in place of internal vector sequences. These results show that XMRV can generate acutely transforming viruses at a low rate, as is typical of other replication-competent retroviruses, and reveal the potential for transforming virus contamination of retroviral vectors made from transformed cell lines.XMRV (xenotropic murine leukemia virus-related virus) has been associated with prostate cancer (19, 20) and chronic fatigue syndrome (12), although some researchers fail to detect XMRV in other populations with these diseases (4, 8). XMRV is found integrated into human genomic DNA from prostate cancer samples, indicating that it is indeed a human retrovirus and not a laboratory contaminant (3, 9). Because of the potential role of XMRV in prostate cancer, we previously tested XMRV for transforming activity in fibroblast and epithelial cell lines. Although XMRV is a simple retrovirus that does not carry a host-derived oncogene, there is precedence for transformation by retroviral Env genes (21, 22). However, transfection of XMRV proviral DNA or viral envelope expression vectors into 208F rat fibroblasts did not result in transformation, and infection of most cell types tested with XMRV did not induce transformation (13). In contrast, infection of 208F cells with XMRV did result in rare transformed foci suggestive of oncogene activation by XMRV. Characterization of cells from three transformed foci produced by infection of 208F cells with virus from HTX cells (a pseudodiploid subclone of HT-1080 fibrosarcoma cells [18]) infected with XMRV and the LAPSN retroviral vector (included as a marker for infection) revealed that all produced XMRV and that one produced a highly active transforming virus (13).     

The AKV Murine Leukemia Virus Is Restricted and Hypermutated by Mouse APOBEC3

APOBEC3 proteins are potent restriction factors against retroviral infection in primates. This restriction is accompanied by hypermutations in the retroviral genome that are attributable to the cytidine deaminase activity of the APOBEC3 proteins. Studies of nucleotide sequence diversity among endogenous gammaretroviruses suggest that the evolution of endogenous retroelements could have been shaped by the mutagenic cytidine deaminase activity of APOBEC3. In mice, however, APOBEC3 appears to restrict exogenous murine retroviruses in the absence of detectable levels of deamination. AKV is an endogenous retrovirus that is involved in causing a high incidence of thymic lymphoma in AKR mice. A comparative analysis of several mouse strains revealed a relatively low level of APOBEC3 expression in AKR mice. Here we show that endogenous mouse APOBEC3 restricts AKV infection and that this restriction likely reflects polymorphisms affecting APOBEC3 abundance rather than differences in the APOBEC3 isoforms expressed. We also observe that restriction of AKV by APOBEC3 is accompanied by G→A hypermutations in the viral genome. Our findings demonstrate that APOBEC3 acts as a restriction factor in rodents affecting the strain tropism of AKV, and they provide good support for the proposal that APOBEC3-mediated hypermutation contributed to the evolution of endogenous rodent retroviral genomes.Viruses that are restrained to infect only a specific animal species, subspecies, or strain have acquired particular features that enable them to circumvent the immune defenses of that particular host. Conversely, the natural hosts for these pathogens are alive today because they have evolved strategies to restrain the infectivities of their own pathogens. A virus with a broad host tropism will typically have evolved under selective pressure from several host factors that it will have encountered and successfully evaded. Ecotropic murine retroviruses generally have a restricted host range, due not only to the limited availability of their cellular receptor, mCAT-1 (58), but also to the various intrinsic restriction factors present in a specific host (7). Fv1 and Fv4 are the expression products of defective endogenous retroviruses that are present as germ line integrations and can interfere with and even block the infectivities of ecotropic retroviruses (6, 25).Mouse APOBEC3 is another type of host-encoded intrinsic restriction factor that can display deoxycytidine deaminase activity on single-stranded DNA (16, 54). APOBEC3 proteins have a potent inhibitory effect on retroelements ranging from primate lentiviruses to murine retrotransposons (reviewed in reference 17). In humans and primates there are seven APOBEC3 genes, most of which have been proposed to act as restriction elements for viruses and retroelements. The most extensively characterized of the primate APOBEC3 proteins are APOBEC3F and APOBEC3G, which constitute powerful restriction factors for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) (reviewed in reference 17). The evidence that these lentiviruses are targets for APOBEC3 action does not come just from in vitro experiments: tissue samples from HIV type 1 (HIV-1)-infected humans contain retroviral sequences exhibiting a pattern of G→A hypermutation that is characteristic of APOBEC3F/G-dependent deoxycytidine deamination (4, 12, 26, 56, 57).In contrast to primates, mice have only a single APOBEC3 gene. This murine APOBEC3 has been shown to be able to inhibit retrotransposition of mouse MusD and intracisternal A particle elements in cotransfection assays (22, 23). However, the lack of any obvious signs of disease, developmental defect, or infertility in APOBEC3-deficient mice indicates that APOBEC3 may not play an essential role in suppressing the transposition of endogenous retroelements in laboratory mice (38, 40). With regard to exogenous retroviruses, mouse APOBEC3 has been shown to hinder the in vivo infectivity of the betaretrovirus mouse mammary tumor virus (MMTV) as well as that of the gammaretrovirus Friend murine leukemia virus (MLV) (40, 55); its activity against Moloney MLV (MoMLV), another gammaretrovirus, is apparently considerably weaker—likely reflecting the fact that MoMLV may have found ways to avoid APOBEC3-mediated restriction (14, 34, 46, 61). In none of these cases, however, does it appear that mouse APOBEC3 hypermutates the retroviral replication intermediates, suggesting that deamination is not central to its mechanism of restricting these retroviruses. Notwithstanding this failure to observe hypermutation of mouse retroviruses by mouse APOBEC3, recent studies of nucleotide sequence diversity among endogenous gammaretroviruses have suggested that the evolution of endogenous retroelements has been shaped by the mutagenic cytidine deaminase activity of APOBEC3 (28, 42). Thus, the picture which emerges is that APOBEC3 acts as one of several restriction factors of mouse retroelements, with some viruses having found ways to avoid APOBEC3-mediated restriction.Different mouse strains exhibit different patterns of APOBEC3 expression (41, 47, 55). Thus, two major mouse APOBEC3 alleles have been identified: one encodes a protein whose sequence is similar to that of the allele expressed in C57BL/6 mice, and the other resembles that of BALB/c mice (47, 55). Two major splicing isoforms of APOBEC3, which either do or do not include exon 5, have also been detected: the relative abundance of these two isoforms differs between strains (36, 41, 47, 55). The restriction of Friend MLV and that of MMTV both appear to be dependent on the identity of the mouse strain, and it has been proposed that this reflects the polymorphism in the sequence and splicing isoforms of APOBEC3 (41, 47, 55).In the course of our work on mouse APOBEC3, we discovered that APOBEC3 was expressed only at a low level in AKR mice. The AKR mouse strain harbors several germ line insertions of an endogenous ecotropic MLV designated AKV, which belongs to the gammaretrovirus family (5, 15, 27, 44, 45). A complex set of recombination events between AKV and nonecotropic endogenous retroviruses results in the production of leukemogenic mink cell focus-inducing viruses that are responsible for inducing a lethal form of thymic lymphoma of T-cell origin in these mice (19, 53). We were interested in determining whether the susceptibility of AKR mice to AKV infection could in part be explained by a failure of the APOBEC3 allele expressed in AKR mice to restrict this virus.Here we show that endogenous murine APOBEC3 in C57BL/6 mice not only acts to restrict AKV infection but also hypermutates AKV replication intermediates, likely providing a powerful block to natural transmission of the virus between mouse strains. We find that the different isoforms of APOBEC3 (whether or not they include exon 5) are effective in AKV restriction and that the differential resistance of lymphocytes from different mouse strains/mutants to AKV infection correlates with the abundance of endogenous APOBEC3 mRNA. Our results indicate that APOBEC3 confers effective protection against germ line integration of retroviral pathogens in rodents, and they provide tangible support to the proposal that DNA editing by APOBEC3 may have participated in the evolution of endogenous retroviral genomes.     

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.     

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.     

The Prostate Cancer-Associated Human Retrovirus XMRV Lacks Direct Transforming Activity but Can Induce Low Rates of Transformation in Cultured Cells

The human retrovirus XMRV (xenotropic murine leukemia virus-related virus) is associated with prostate cancer, but a causal relationship has not been established. Here, we have used cultured fibroblast and epithelial cell lines to test the hypothesis that XMRV might have direct transforming activity but found only rare transformation events, suggestive of indirect transformation, even when the target cells expressed the human Xpr1 cell entry receptor for XMRV. Characterization of cells from three transformed foci showed that all were infected with and produced XMRV, and one produced a highly active transforming virus, presumably generated by recombination between XMRV and host cell nucleic acids. Given the sequence similarity of XMRV to mink cell focus-forming (MCF) viruses and the enhanced leukemogenic activity of the latter, we tested XMRV for related MCF-like cytopathic activities in cultured mink cells but found none. These results indicate that XMRV has no direct transforming activity but can activate endogenous oncogenes, resulting in cell transformation. As part of these experiments, we show that XMRV can infect and be produced at a high titer from human HT-1080 fibrosarcoma cells that express TRIM5α (Ref1), showing that XMRV is resistant to TRIM5α restriction. In addition, XMRV poorly infects NIH 3T3 cells expressing human Xpr1 but relatively efficiently infects BALB 3T3 cells expressing human Xpr1, showing that XMRV is a B-tropic virus and that its infectivity is regulated by the Fv1 mouse locus.The association of human prostate cancer with mutations that impair the function of the antiviral defense protein RNase L suggested a role for virus in prostate cancer. Indeed, analysis of cDNA from prostate tumors by use of a DNA microarray (Virochip) containing conserved DNA sequences from all known virus families indicated the presence of a novel gammaretrovirus in 40% of prostate cancer patients having homozygous R462Q mutations in RNase L (35). Cloning and sequencing of the virus revealed a close similarity to mouse xenotropic retroviruses; thus, the new virus was named XMRV (xenotropic murine leukemia virus-related virus) (35). Importantly, XMRV has been found integrated into human genomic DNA from tumor-bearing prostatic tissue samples of 11 patients, showing that XMRV can indeed infect humans and is not a laboratory contaminant (7, 13). Although an initial study found XMRV only in tumor stromal cells (35), recent studies have found XMRV in the prostate carcinoma cell line 22Rv1 (14) and in malignant epithelial cells in prostate tumors (34).XMRV lacks a host cell-derived oncogene, but examples of oncogenic activity in Env proteins from other retroviruses (1, 6, 16, 24) raise the possibility that the Env protein of XMRV might also be oncogenic. Such activity could be a result of interaction of the XMRV Env protein with the virus entry receptor Xpr1 (7, 14), which shows similarity to a yeast protein involved in G protein-coupled signal transduction (2), or interaction with other cellular proteins that do not function as virus entry receptors, as is the case for jaagsiekte sheep retrovirus (JSRV) Env (interacting protein unknown) (16) and the Env protein of spleen focus-forming virus, which interacts with and activates the erythropoietin receptor and the receptor tyrosine kinase Stk (24). Detection of XMRV oncogenic activity would strengthen the argument for a role for XMRV in prostate cancer.In addition, while XMRV shows the highest sequence similarity to the mouse xenotropic retroviruses, it is also similar to the mink cell focus-forming (MCF) retroviruses of mice, which are highly leukemogenic due to their ability to multiply reinfect cells, leading to more-frequent activation of cellular oncogenes (36). MCF viruses were first defined by their ability to induce foci of altered cells in mink cell layers (11). Initially, it was unclear whether these foci were the result of cell transformation or cytopathic effects of the virus (11), but it is clear now that these foci result from cytopathic effects related to the ability of MCF viruses to multiply reinfect cells in what can be a receptor-independent manner, leading to cell apoptosis (23, 36, 37). It was thus important to determine if XMRV has similar properties and might be able to more frequently activate cellular oncogenes.Here, we have found that while XMRV lacks direct transforming activity in the fibroblast and epithelial cell lines tested and does not induce cytopathic effects typical of multiple reinfection by MCF viruses, it is able to induce rare transformed foci in a rat fibroblast cell line. Interestingly, in one case, transformation led to the production of a highly active oncogenic retrovirus.     

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.     

Evaluation of Cellular Determinants Required for In Vitro Xenotropic Murine Leukemia Virus-Related Virus Entry into Human Prostate Cancer and Noncancerous Cells

The newly identified retrovirus—the xenotropic murine leukemia virus-related virus (XMRV)—has recently been shown to be strongly associated with familial prostate cancer in humans (A. Urisman et al., PLoS Pathog. 2:e25, 2006). While that study showed evidence of XMRV infection exclusively in the prostatic stromal fibroblasts, a recent study found XMRV protein antigens mainly in malignant prostate epithelial cells (R. Schlaberg et al., Proc. Natl. Acad. Sci. U. S. A. 106:16351-16356, 2009). To help elucidate the mechanisms behind XMRV infection, we show that prostatic fibroblast cells express Xpr1, a known receptor of XMRV, but its expression is absent in other cell lines of the prostate (i.e., epithelial and stromal smooth muscle cells). We also show that certain amino acid residues located within the predicted extracellular loop (ECL3 and ECL4) sequences of Xpr1 are required for efficient XMRV entry. Although we found strong evidence to support XMRV infection of prostatic fibroblast cell lines via Xpr1, we learned that XMRV was indeed capable of infecting cells that did not necessarily express Xpr1, such as those of the prostatic epithelial and smooth muscle origins. Further studies suggest that the expression of Xpr1 and certain genotypes of the RNASEL gene, which could restrict XMRV infection, may play important roles in defining XMRV tropisms in certain cell types. Collectively, our data reveal important cellular determinants required for XMRV entry into different human prostate cells in vitro, which may provide important insights into the possible role of XMRV as an etiologic agent in human prostate cancer.Prostate cancer is the most common male malignancy in Western countries and the second most common cause of cancer-related deaths in males worldwide (15, 24). The known risk factors for prostate cancer are hormones (i.e., androgens), diet, sex, and race, as well as environmental and genetic factors (27). A recent study suggests that susceptibility to prostate cancer can be influenced by the genetic variations associated with an antagonistic coevolution, which occurs between a specific host locus (RNASEL), known to be involved in antiviral innate immune defense, and a viral pathogen (38). Indeed, several epidemiologic studies have supported the involvement of the RNASEL gene in the prostate cancer etiology (4, 5, 30, 31), whereas other studies do not (9, 22, 34, 43). Some studies have reported that individuals with a single mutated copy of the RNASEL gene have a 50% increased risk for prostate cancer, whereas those with homozygous mutant RNASEL alleles have a 2-fold-increased risk of prostate cancer (5).The RNASEL gene encodes for the RNase L protein, a constitutively expressed latent endoribonuclease, which mediates the interferon-inducible 2-5A system against viral and/or cellular double-stranded RNAs (8, 16, 20, 23, 49, 50). The RNase L “Q” variant allele (R462Q) shows a 3-fold decrease in catalytic activity compared to the wild-type enzyme (5, 44). The possible association of mutant RNASEL alleles with human prostate cancers suggests an enhanced susceptibility of prostate tissues to a viral agent. This hypothesis has led to the recent identification of a new human retrovirus, xenotropic murine leukemia virus (MuLV)-related virus (XMRV), in 40% of prostate cancer patients with the QQ variant alleles of RNASEL compared to 1.5% among heterozygous (RQ) and wild-type (RR) RNASEL carriers (41). XMRV virus infection appears to be susceptible to inhibition by interferon and its downstream effector RNase L protein (7). However, a recent study has provided some evidence to show that XMRV infection is independent of the RNASEL genotype (34), suggesting that population differences and/or other environmental or genetic factors may influence the impact of RNASEL on prostate cancer development.The XMRV genome is 8,185 nucleotides in length and shares up to 95% overall nucleotide sequence identity with known xenotropic MuLVs (41). One receptor for xenotropic MuLVs is Xpr1, a 696-amino-acid protein with multiple transmembrane-spanning domains (2). Expression of this protein in Chinese hamster ovary (CHO) cells that are not known to express Xpr1 endogenously confers an enhanced susceptibility of these cells to xenotropic MuLV infection (2). Infection of hamster and mouse cells with XMRV-like virus that is derived from a prostate cancer cell line (22Rv1) also requires Xpr1 as a receptor (18). Earlier studies have demonstrated the importance of certain residues located within the putative third and fourth extracellular loops (ECL3 and ECL4) of Mus dunni''s Xpr1 in conferring infection by xenotropic MuLVs (25). Furthermore, it has been shown that the specific and common receptor determinants for xenotropic and polytropic murine retroviruses are simultaneously present in discrete domains of a single Xpr1 protein (42). In the present study, we characterized for the first time the important molecular determinants on Xpr1 required for XMRV infection and investigated the role of RNase L in restricting XMRV infection of various human prostate cancer and noncancerous cell lines.