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.     

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.     

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.     

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.     

Dissection of the HIV Vif Interaction with Human E3 Ubiquitin Ligase

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

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.     

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.     

Identification of amino acid residues in HIV-1 Vif critical for binding and exclusion of APOBEC3G/F

To define a region(s) in human immunodeficiency virus type 1 (HIV-1) Vif that involves binding to its target APOBEC3G (A3G), we have generated a series of site-specific proviral vif mutants. Of 30 mutants examined, 15 did not grow at all or grew more poorly than wild-type virus in non-permissive cells. Eight clones with N-terminal mutations located outside of the HCCH motif and BC-box, which are known to be directly crucial for the degradation of A3G, were chosen from these growth-defective mutants and mainly analyzed in detail for functional activity of their mutant Vif proteins. By single-cycle replication and immunoprecipitation/immunoblotting analyses, mutants designated W21A, S32A, W38A, Y40A, and H43A were demonstrated to hardly or poorly bind to and neutralize A3G. Upon transfection, these mutants produced progeny virions containing much more A3G than wild-type clone. Interestingly, while mutants designated E76A and W79A acted normally to inactivate A3G, they were found to exhibit a Vif-defective phenotype against A3F. Another unique mutant designated Y69A incompetent against both of A3G/F was also identified. Our results here have indicated that at least two distinct regions in the N-terminal half of HIV-1 Vif are critical for binding and exclusion of A3G/F.     

The Vif protein of HIV triggers degradation of the human antiretroviral DNA deaminase APOBEC3G

APOBEC3G is a human cellular enzyme that is incorporated into retroviral particles and acts to restrict retroviral replication in infected cells by deaminating dC to dU in the first (minus)-strand cDNA replication intermediate. HIV, however, encodes a protein (virion infectivity factor, Vif ), which overcomes APOBEC3G-mediated restriction but by an unknown mechanism. Here, we show that Vif triggers APOBEC3G degradation by a proteasome-dependent pathway and that an 80 amino acid region of APOBEC3G surrounding its first zinc coordination motif is sufficient to confer the ability to partake in an interaction involving Vif. Inhibitors of this interaction might therefore prove therapeutically useful in blocking Vif-mediated APOBEC3G destruction.     

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.     

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.     

Ubiquitination of APOBEC3G by an HIV-1 Vif-Cullin5-Elongin B-Elongin C complex is essential for Vif function

The human immunodeficiency virus type 1 (HIV-1) virion infectivity factor (Vif) overcomes the antiviral activity of APOBEC3G to protect HIV-1 DNA from G-to-A hypermutation. Vif targets APOBEC3G for ubiquitination and proteasomal degradation by forming an SCF-like E3 ubiquitin ligase complex composed of Cullin5, Elongin B, and Elongin C (Vif-BC-Cul5) through a novel SOCS-box motif. In this paper, we have established an in vitro ubiquitin conjugation assay with purified Vif-BC-Cul5 complex and reported that the Vif-BC-Cul5 complex could function as an E3 ligase for APOBEC3G in vitro. A Vif-BC-Cul5 complex promotes the in vitro ubiquitination of the wild type, APOBEC3G but not that of D128K mutant, which does not interact with Vif. We have also investigated several loss-of-function Vif mutants. One mutant, SLQ144/146AAA, lost its activity on APOBEC3G because it could not form a complex due to mutations in SOCS-box motif. Other mutants, C114S and C133S, also lost their activity because of loss of the E3 ligase activity of a Vif-BC-Cul5 complex, although these mutants retained the ability to bind to APOBEC3G as well as Cul5 complex. These findings suggest that the E3 ubiquitin ligase activity of the Vif-BC-Cul5 complex is essential for Vif function against APOBEC3G.     

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.     

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.     

Regulation of Apobec3F and human immunodeficiency virus type 1 Vif by Vif-Cul5-ElonB/C E3 ubiquitin ligase

The human cytidine deaminase Apobec3F (h-A3F), a protein related to the previously recognized antiviral factor Apobec3G (h-A3G), has antiviral activity against human immunodeficiency virus type 1 (HIV-1) that is suppressed by the viral protein Vif. The mechanism of HIV-1 Vif-mediated suppression of h-A3F is not fully understood. Here, we demonstrate that while h-A3F, like h-A3G, was able to suppress primate lentiviruses other than HIV-1 (simian immunodeficiency virus from African green monkeys [SIVagm] and Rhesus macaques [SIVmac]), the interaction between Vif proteins and h-A3F appeared to differ from that with h-A3G. H-A3F showed no change in its species specificity against HIV-1 or SIVagm Vif when a negatively charged amino acid was replaced with a lysine at position 128, a residue critical for h-A3G recognition by HIV-1 Vif. However, HIV-1 Vif, but not SIVagm Vif, was able to bind h-A3F and induce its polyubiquitination and degradation through the Cul5-containing E3 ubiquitin ligase. Interference with Cul5-E3 ligase function by depletion of Cul5, through RNA interference or overexpression of Cul5 mutants, blocked the ability of HIV-1 Vif to suppress h-A3F. A BC-box mutant of HIV-1 Vif that failed to recruit Cul5-E3 ligase but was still able to interact with h-A3F failed to suppress h-A3F. Interestingly, interference with Cul5-E3 ligase function or overexpression of h-A3F or h-A3G also increased the stability of HIV-1 Vif, suggesting that like the substrate molecules h-A3F and h-A3G, the substrate receptor protein Vif is itself also regulated by Cul5-E3 ligase. Our results indicate that Cul5-E3 ligase appears to be a common pathway hijacked by HIV-1 Vif to defeat both h-A3F and h-A3G. Developing inhibitors to disrupt the interaction between Vif and Cul5-E3 ligase could be therapeutically useful, allowing multiple host antiviral factors to suppress HIV-1.     

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.     

On the Solution Conformation and Dynamics of the HIV-1 Viral Infectivity Factor

Human immunodeficiency virus-1 (HIV-1) has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host cell enzymes. HIV-1 Vif [viral (also called virion) infectivity factor], one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and downregulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of the protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method that is well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation, suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion, indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution, including the APOBEC3G/F binding site and the HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif.