Insight into the HIV-1 Vif SOCS-box–ElonginBC interaction

The HIV-1 viral infectivity factor (Vif) neutralizes cell-encoded antiviral APOBEC3 proteins by recruiting a cellular ElonginB (EloB)/ElonginC (EloC)/Cullin5-containing ubiquitin ligase complex, resulting in APOBEC3 ubiquitination and proteolysis. The suppressors-of-cytokine-signalling-like domain (SOCS-box) of HIV-1 Vif is essential for E3 ligase engagement, and contains a BC box as well as an unusual proline-rich motif. Here, we report the NMR solution structure of the Vif SOCS–ElonginBC (EloBC) complex. In contrast to SOCS-boxes described in other proteins, the HIV-1 Vif SOCS-box contains only one α-helical domain followed by a β-sheet fold. The SOCS-box of Vif binds primarily to EloC by hydrophobic interactions. The functionally essential proline-rich motif mediates a direct but weak interaction with residues 101–104 of EloB, inducing a conformational change from an unstructured state to a structured state. The structure of the complex and biophysical studies provide detailed insight into the function of Vif''s proline-rich motif and reveal novel dynamic information on the Vif–EloBC interaction.     

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

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

Hydrodynamic and functional analysis of HIV-1 Vif oligomerization

HIV-1 Vif is an accessory protein that induces the proteasomal degradation of the host restriction factor, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G). The N-terminal half of Vif binds to APOBEC3G, and the C-terminal half binds to subunits of a cullin 5-based ubiquitin ligase. This Vif-directed ubiquitin ligase induces the degradation of APOBEC3G (a cytidine deaminase) and thereby protects the viral genome from mutation. A conserved PPLP motif near the C-terminus of Vif is essential for Vif function and is also involved in Vif oligomerization. However, the mechanism and functional significance of Vif oligomerization is unclear. We employed analytical ultracentrifugation to examine the oligomeric properties of Vif in solution. Contrary to previous reports, we find that Vif oligomerization does not require the conserved PPLP motif. Instead, our data suggest a more complex mechanism involving interactions among the HCCH motif, the BC box, and downstream residues in Vif. Mutation of residues near the PPLP motif (S165 and V166) affected the oligomeric properties of Vif and weakened the ability of Vif to bind and induce the degradation of APOBEC3G. We propose that Vif oligomerization may represent a mechanism for regulating interactions with APOBEC3G.     

Vif overcomes the innate antiviral activity of APOBEC3G by promoting its degradation in the ubiquitin-proteasome pathway

Viruses must overcome diverse intracellular defense mechanisms to establish infection. The Vif (virion infectivity factor) protein of human immunodeficiency virus 1 (HIV-1) acts by overcoming the antiviral activity of APOBEC3G (CEM15), a cytidine deaminase that induces G to A hypermutation in newly synthesized viral DNA. In the absence of Vif, APOBEC3G incorporation into virions renders HIV-1 non-infectious. We report here that Vif counteracts the antiviral activity of APOBEC3G by targeting it for destruction by the ubiquitin-proteasome pathway. Vif forms a complex with APOBEC3G and enhances APOBEC3G ubiquitination, resulting in reduced steady-state APOBEC3G levels and a decrease in protein half-life. Furthermore, Vif-dependent degradation of APOBEC3G is blocked by proteasome inhibitors or ubiquitin mutant K48R. A mutation of highly conserved cysteines or the deletion of a conserved SLQ(Y/F)LA motif in Vif results in mutants that fail to induce APOBEC3G degradation and produce non-infectious HIV-1; however, mutations of conserved phosphorylation sites in Vif that impair viral replication do not affect APOBEC3G degradation, suggesting that Vif is important for other functions in addition to inducing proteasomal degradation of APOBEC3G. Vif is monoubiquitinated in the absence of APOBEC3G but is polyubiquitinated and rapidly degraded when APOBEC3G is coexpressed, suggesting that coexpression accelerates the degradation of both proteins. These results suggest that Vif functions by targeting APOBEC3G for degradation via the ubiquitin-proteasome pathway and implicate the proteasome as a site of dynamic interplay between microbial and cellular defenses.     

Encapsidation of APOBEC3G into HIV-1 virions involves lipid raft association and does not correlate with APOBEC3G oligomerization

The human immunodeficiency virus type 1 (HIV-1) Vif plays a crucial role in the viral life cycle by antagonizing a host restriction factor APOBEC3G (A3G). Vif interacts with A3G and induces its polyubiquitination and subsequent degradation via the formation of active ubiquitin ligase (E3) complex with Cullin5-ElonginB/C. Although Vif itself is also ubiquitinated and degraded rapidly in infected cells, precise roles and mechanisms of Vif ubiquitination are largely unknown. Here we report that MDM2, known as an E3 ligase for p53, is a novel E3 ligase for Vif and induces polyubiquitination and degradation of Vif. We also show the mechanisms by which MDM2 only targets Vif, but not A3G that binds to Vif. MDM2 reduces cellular Vif levels and reversely increases A3G levels, because the interaction between MDM2 and Vif precludes A3G from binding to Vif. Furthermore, we demonstrate that MDM2 negatively regulates HIV-1 replication in non-permissive target cells through Vif degradation. These data suggest that MDM2 is a regulator of HIV-1 replication and might be a novel therapeutic target for anti-HIV-1 drug.     

Characterization of conserved motifs in HIV-1 Vif required for APOBEC3G and APOBEC3F interaction

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G, or A3G) and related cytidine deaminases such as apolipoprotein B mRNA-editing catalytic polypeptide-like 3F (APOBEC3F, or A3F) are potent inhibitors of retroviruses. Formation of infectious human immunodeficiency virus (HIV)-1 requires suppression of multiple cytidine deaminases by Vif. HIV-1 Vif suppresses various APOBEC3 proteins through a common mechanism by recruiting Cullin5, ElonginB, and ElonginC E3 ubiquitin ligase to induce target protein polyubiquitination and proteasome-mediated degradation. Domains in Vif that mediate APOBEC3 recognition have not been fully characterized. In the present study, we identified a VxIPLx_4-5LxΦx₂YWxL motif in HIV-1 Vif, which is required for efficient interaction between Vif and A3G, Vif-mediated A3G degradation and virion exclusion, and functional suppression of the A3G antiviral activity. Amino acids 52 to 72 of HIV-1 Vif (including the VxIPLx_4-5LxΦx₂YWxL motif) alone could mediate interaction with A3G, and this interaction was abolished by mutations of two hydrophobic amino acids in this region. We have also observed that a Vif mutant was ineffective against A3G, yet it retained the ability to interact with Cullin5-E3 ubiquitin complex and A3G, suggesting that interaction with A3G is necessary but not sufficient to inhibit its antiviral function. Unlike the previously identified motif of HIV-1 Vif amino acids 40 to 44, which is only important for A3G suppression, the VxIPLx_4-5LxΦx₂YWxL motif is also required for efficient A3F interaction and suppression. On the other hand, another motif, TGERxW, of HIV-1 Vif amino acids 74 to 79 was found to be mainly important for A3F interaction and inhibition. Both the VxIPLx_4-5LxΦx₂YWxL and TGERxW motifs are highly conserved among HIV-1, HIV-2, and various simian immunodeficiency virus Vif proteins. Our data suggest that primate lentiviral Vif molecules recognize their autologous APOBEC3 proteins through conserved structural features that represent attractive targets for the development of novel inhibitors.     

Human immunodeficiency virus type 1 Vif induces cell cycle delay via recruitment of the same E3 ubiquitin ligase complex that targets APOBEC3 proteins for degradation

Human immunodeficiency virus type 1 (HIV-1) Vif recruits a Cullin 5 ubiquitin ligase that targets APOBEC3 proteins for degradation. Recently, Vif has also been shown to induce cell cycle disturbance in G(2). We show that in contrast to the expression of Vpr, the expression of Vif does not preclude cell division, and therefore, Vif causes delay and not arrest in G(2). We also demonstrate that the interaction of Vif with the ubiquitin ligase is required for cell cycle disruption, as was previously shown for HIV-1 Vpr. The presence of APOBEC3 D/E, F, and G had no influence on Vif-induced alteration of the cell cycle. We conclude that cell cycle delay by Vif is a result of ubiquitination and degradation of a cellular protein that is different from the known APOBEC3 family members.     

Characterization of the interaction of full-length HIV-1 Vif protein with its key regulator CBFβ and CRL5 E3 ubiquitin ligase components

Human immunodeficiency virus-1 (HIV-1) viral infectivity factor (Vif) is essential for viral replication because of its ability to eliminate the host's antiviral response to HIV-1 that is mediated by the APOBEC3 family of cellular cytidine deaminases. Vif targets these proteins, including APOBEC3G, for polyubiquitination and subsequent proteasome-mediated degradation via the formation of a Cullin5-ElonginB/C-based E3 ubiquitin ligase. Determining how the cellular components of this E3 ligase complex interact with Vif is critical to the intelligent design of new antiviral drugs. However, structural studies of Vif, both alone and in complex with cellular partners, have been hampered by an inability to express soluble full-length Vif protein. Here we demonstrate that a newly identified host regulator of Vif, core-binding factor-beta (CBFβ), interacts directly with Vif, including various isoforms and a truncated form of this regulator. In addition, carboxyl-terminal truncations of Vif lacking the BC-box and cullin box motifs were sufficient for CBFβ interaction. Furthermore, association of Vif with CBFβ, alone or in combination with Elongin B/C (EloB/C), greatly increased the solubility of full-length Vif. Finally, a stable complex containing Vif-CBFβ-EloB/C was purified in large quantity and shown to bind purified Cullin5 (Cul5). This efficient strategy for purifying Vif-Cul5-CBFβ-EloB/C complexes will facilitate future structural and biochemical studies of Vif function and may provide the basis for useful screening approaches for identifying novel anti-HIV drug candidates.     

Identification of an APOBEC3G binding site in human immunodeficiency virus type 1 Vif and inhibitors of Vif-APOBEC3G binding

The APOBEC3 cytidine deaminases are potent antiviral factors that restrict replication of human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif binds APOBEC3G and APOBEC3F and targets these proteins for ubiquitination by forming an E3 ubiquitin ligase with cullin 5 and elongins B and C. The N-terminal region of Vif is required for APOBEC3G binding, but the binding site(s) is unknown. To identify the APOBEC3G binding site in Vif, we established a scalable binding assay in a format compatible with development of high-throughput screens. In vitro binding assays using recombinant proteins identified Vif peptides and monoclonal antibodies that inhibit Vif-APOBEC3G binding and suggested involvement of Vif residues 33 to 83 in APOBEC3G binding. Cell-based binding assays confirmed these results and demonstrated that residues 40 to 71 in the N terminus of Vif contain a nonlinear binding site for APOBEC3G. Mutation of the highly conserved residues His42/43 but not other charged residues in this region inhibited Vif-APOBEC3G binding, Vif-mediated degradation of APOBEC3G, and viral infectivity. In contrast, mutation of these residues had no significant effect on Vif binding and degradation of APOBEC3F, suggesting a differential requirement for His42/43 in Vif binding to APOBEC3G and APOBEC3F. These results identify a nonlinear APOBEC3 binding site in the N terminus of Vif and demonstrate that peptides or antibodies directed against this region can inhibit Vif-APOBEC3G binding, validating the Vif-APOBEC3 interface as a potential drug target.     

The SOCS-Box of HIV-1 Vif Interacts with ElonginBC by Induced-Folding to Recruit Its Cul5-Containing Ubiquitin Ligase Complex

The HIV-1 viral infectivity factor (Vif) protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC), cullin 5 (Cul5) and RING-box 2 (Rbx2), to the anti-viral proteins APOBEC3G (A3G) and APOBEC3F (A3F) and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif''s BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function.     

Evolutionarily conserved pressure for the existence of distinct G2/M cell cycle arrest and A3H inactivation functions in HIV-1 Vif

HIV-1 Vif assembles the Cul5-EloB/C E3 ubiquitin ligase to induce proteasomal degradation of the cellular antiviral APOBEC3 proteins. Detailed structural studies have confirmed critical functional domains in Vif that we have previously identified as important for the interaction of EloB/C, Cul5, and CBFβ. However, the mechanism by which Vif recognizes substrates remains poorly understood. Specific regions of Vif have been identified as being responsible for binding and depleting APOBEC3G and APOBEC3F. Interestingly, we have now identified distinct yet overlapping domains that are required for HIV-1 Vif-mediated G2/M-phase cell cycle arrest and APOBEC3H degradation, but not for the inactivation of APOBEC3G or APOBEC3F. Surprisingly, Vif molecules from primary HIV-1 variants that caused G2/M arrest were unable to inactivate APOBEC3H; on the other hand, HIV-1 Vif variants that could inactivate APOBEC3H were unable to induce G2/M arrest. All of these Vif variants still maintained the ability to inactivate APOBEC3G/F. Thus, primary HIV-1 variants have evolved to possess distinct functional activities that allow them to suppress APOBEC3H or cause G2 cell cycle arrest, using mutually exclusive interface domains. APOBEC3H depletion and G2 arrest are apparently evolutionary selected features that cannot co-exist on a single Vif molecule. The existence and persistence of both types of HIV-1 Vif variant suggests the importance of APOBEC3H suppression and cell cycle regulation for HIV-1''s survival in vivo.     

Muf1, a novel Elongin BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1 to reconstitute a ubiquitin ligase

The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.     

Molecular Insight into the Conformational Dynamics of the Elongin BC Complex and Its Interaction with HIV-1 Vif

The human immunodeficiency virus type 1 virion infectivity factor (Vif) inhibits the innate viral immunity afforded by the APOBEC3 family of cytidine deaminases. Vif targets the APOBEC3 family for poly-ubiquitination and subsequent proteasomal degradation by linking the Elongin-BC-dependent ubiquitin ligase complex with the APOBEC3 proteins. The interaction between Vif and the heterodimeric Elongin BC complex, which is mediated by Vif's viral suppressor of cytokine signaling box, is essential for Vif function. The biophysical consequences of the full-length Vif:Elongin BC interaction have not been extensively reported. In this study, hydrogen exchange mass spectrometry was used to dissect the Vif:Elongin BC interaction. Elongin C was found to be highly dynamic in the Elongin BC complex while Elongin B was much more stable. Recombinant full-length Vif interacted with the Elongin BC complex in vitro with a K_d of 1.9 μM and resulted in observable changes in deuterium uptake in both Elongin C and B. Upon binding to Elongin BC, no significant global conformational changes were detected in Vif by hydrogen exchange mass spectrometry, but a short fragment of Vif that consisted of the viral suppressor of cytokine signaling box showed decreased deuterium incorporation upon Elongin BC incubation, suggesting that this region folds upon binding.     

Structural insight into the human immunodeficiency virus Vif SOCS box and its role in human E3 ubiquitin ligase assembly

Human immunodeficiency virus (HIV) virion infectivity factor (Vif) causes the proteasome-mediated destruction of human antiviral protein APOBEC3G by tethering it to a cellular E3 ubiquitin ligase composed of ElonginB, ElonginC, Cullin5, and Rbx2. It has been proposed that HIV Vif hijacks the E3 ligase through two regions within its C-terminal domain: a BC box region that interacts with ElonginC and a novel zinc finger motif that interacts with Cullin5. We have determined the crystal structure of the HIV Vif BC box in complex with human ElonginB and ElonginC. This complex presents direct structural evidence of the recruitment of a human ubiquitin ligase by a viral BC box protein that mimics the conserved interactions of cellular ubiquitin ligases. We further mutated conserved hydrophobic residues in a region downstream of the Vif BC box. These mutations demonstrate that this region, the Vif Cullin box, composes a third E3-ligase recruiting site critical for interaction between Vif and Cullin5. Furthermore, our homology modeling reveals that the Vif Cullin box and zinc finger motif may be positioned adjacent to the N terminus of Cullin5 for interaction with loop regions in the first cullin repeat of Cullin5.     

A zinc-binding region in Vif binds Cul5 and determines cullin selection

Human immunodeficiency virus-1 (HIV-1) Vif overcomes the anti-viral activity of APOBEC3G by targeting it for ubiquitination via a Cullin 5-ElonginB-ElonginC (Cul5-EloBC) E3 ligase. Vif associates with Cul5-EloBC through a BC-box motif that binds EloC, but the mechanism by which Vif selectively recruits Cul5 is poorly understood. Here we report that a region of Vif (residues 100-142) upstream of the BC-box binds selectively to Cul5 in the absence of EloC. This region contains a zinc coordination site HX5CX17-18CX3-5H (HCCH), with His/Cys residues at positions 108, 114, 133, and 139 coordinating one zinc ion. The HCCH zinc coordination site, which is conserved among primate lentivirus Vif proteins, does not correspond to any known class of zinc-binding motif. Mutations of His/Cys residues in the HCCH motif impair zinc coordination, Cul5 binding, and APOBEC3G degradation. Mutations of conserved hydrophobic residues (Ile-120, Ala-123, and Leu-124) located between the two Cys residues in the HCCH motif disrupt binding of the zinc-coordinating region to Cul5 and inhibit APOBEC3G degradation. The Vif binding site maps to the first cullin repeat in the N terminus of Cul5. These data suggest that the zinc-binding region in Vif is a novel cullin interaction domain that mediates selective binding to Cul5. We propose that the HCCH zinc-binding motif facilitates Vif-Cul5 binding by playing a structural role in positioning hydrophobic residues for direct contact with Cul5.     

Characterization of a novel Cullin5 binding domain in HIV-1 Vif

Human immunodeficiency virus tyoe 1 (HIV-1) Vif counteracts host restriction cytidine deaminase (APOBEC3G) A3G by co-opting the cellular ubiquitin-proteasome machinery. Vif utilizes a viral-specific BC-box to recruit ElonginB-ElonginC and a novel zinc-binding HCCH motif to recruit Cullin5 (Cul5) to form an E3 ubiquitin ligase targeting A3G for polyubiquitination and subsequently proteasomal degradation. To determine the structural requirements in HIV-1 Vif HCCH motif for Cul5 binding and Vif function, we investigated the arrangement of the His and Cys residues, the role of the spacing between them, and the requirement for the conserved residues. Our data demonstrate that exchanging Cys for His and vice versa in the highly conserved Zn-coordinating HCCH motif disrupted Vif function and interaction with Cul5. Moreover, the maintenance of both conserved residues and spacing within the HCCH motif is critical for Vif function. We have identified a "viral Cul5 box" with consensus Hx2YFxCFx4Phix2APhix7-8Cx5H that is required for Cul5 selection and subsequent A3G degradation. This novel motif may represent a potential new target for anti-viral drug development.     

Small-molecule inhibition of human immunodeficiency virus type 1 replication by targeting the interaction between Vif and ElonginC

The HIV-1 viral infectivity factor (Vif) protein is essential for viral replication. Vif recruits cellular ElonginB/C-Cullin5 E3 ubiquitin ligase to target the host antiviral protein APOBEC3G (A3G) for proteasomal degradation. In the absence of Vif, A3G is packaged into budding HIV-1 virions and introduces multiple mutations in the newly synthesized minus-strand viral DNA to restrict virus replication. Thus, the A3G-Vif-E3 complex represents an attractive target for development of novel anti-HIV drugs. In this study, we identified a potent small molecular compound (VEC-5) by virtual screening and validated its anti-Vif activity through biochemical analysis. We show that VEC-5 inhibits virus replication only in A3G-positive cells. Treatment with VEC-5 increased cellular A3G levels when Vif was coexpressed and enhanced A3G incorporation into HIV-1 virions to reduce viral infectivity. Coimmunoprecipitation and computational analysis further attributed the anti-Vif activity of VEC-5 to the inhibition of Vif from direct binding to the ElonginC protein. These findings support the notion that suppressing Vif function can liberate A3G to carry out its antiviral activity and demonstrate that regulation of the Vif-ElonginC interaction is a novel target for small-molecule inhibitors of HIV-1.     

Identification of a Cullin5-ElonginB-ElonginC E3 complex in degradation of feline immunodeficiency virus Vif-mediated feline APOBEC3 proteins

Various feline APOBEC3 (fA3) proteins exhibit broad antiviral activities against a wide range of viruses, such as feline immunodeficiency virus (FIV), feline foamy virus (FFV), and feline leukemia virus (FeLV), as well as those of other species. This activity can be counteracted by the FIV Vif protein, but the mechanism by which FIV Vif suppresses fA3s is unknown. In the present study, we demonstrated that FIV Vif could act via a proteasome-dependent pathway to overcome fA3s. FIV Vif interacted with feline cellular proteins Cullin5 (Cul5), ElonginB, and ElonginC to form an E3 complex to induce degradation of fA3s. Both the dominant-negative Cul5 mutant and a C-terminal hydrophilic replacement ElonginC mutant potently disrupted the FIV Vif activity against fA3s. Furthermore, we identified a BC-box motif in FIV Vif that was essential for the recruitment of E3 ubiquitin ligase and also required for FIV Vif-mediated degradation of fA3s. Moreover, despite the lack of either a Cul5-box or a HCCH zinc-binding motif, FIV Vif specifically selected Cul5. Therefore, FIV Vif may interact with Cul5 via a novel mechanism. These finding imply that SOCS proteins may possess distinct mechanisms to bind Cul5 during formation of the Elongin-Cullin-SOCS box complex.