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.     

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.     

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.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

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.