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 multimerization of human immunodeficiency virus type I Vif protein: a requirement for Vif function in the viral life cycle

The Vif (virion infectivity factor protein of human immunodeficiency virus type I (HIV-1) is essential for viral replication in vivo and productive infection of peripheral blood mononuclear cells, macrophages, and H9 T-cells. However, the molecular mechanism(s) of Vif remains unknown and needs to be further determined. In this report, we show that, like many other proteins encoded by HIV-1, Vif proteins possess a strong tendency toward self-association. In relatively native conditions, Vif proteins formed multimers in vitro, including dimers, trimers, or tetramers. Through in vivo binding assays such as coimmunoprecipitation and the mammalian two-hybrid system, we also demonstrated that Vif proteins could interact with each other within a cell, indicating that the multimerization of Vif proteins is not simply due to fortuitous aggregation. Further studies indicated that the domain affecting Vif self-association is located at the C terminus of this protein, especially the proline-enriched 151-164 region. Moreover, we found that a Vif mutant with deletion at amino acid 151-164 was unable to rescue the infectivity of vif-defective viruses generated from H9 T-cells, suggesting that the multimerization of Vif proteins could be important for Vif function in the viral life cycle. Our studies identified a new feature of Vif and should accelerate our understanding of its role in HIV-1 pathogenesis.     

Functional analysis of Vif protein shows less restriction of human immunodeficiency virus type 2 by APOBEC3G

Viral infectivity factor (Vif) is one of the human immunodeficiency virus (HIV) accessory proteins and is conserved in the primate lentivirus group. This protein is essential for viral replication in vivo and for productive infection of nonpermissive cells, such as peripheral blood mononuclear cells (PBMC). Vif counteracts an antiretroviral cellular factor in nonpermissive cells named CEM15/APOBEC3G. Although HIV type 1 (HIV-1) Vif protein (Vif1) can be functionally replaced by HIV-2 Vif protein (Vif2), its identity is very small. Most of the functional studies have been carried out with Vif1. Characterization of functional domains of Vif2 may elucidate its function, as well as differences between HIV-1 and HIV-2 infectivity. Our aim was to identify the permissivity of different cell lines for HIV-2 vif-minus viruses. By mutagenesis specific conserved motifs of HIV-2 Vif protein were analyzed, as well as in conserved motifs between Vif1 and Vif2 proteins. Vif2 mutants were examined for their stability, expression, and cellular localization in order to characterize essential domains of Vif2 proteins. Viral replication in various target cells (PBMC and H9, A3.01, U38, and Jurkat cells) and infectivity in single cycle assays in the presence of APOBEC3G were also analyzed. Our results of viral replication show that only PBMC have a nonpermissive phenotype in the absence of Vif2. Moreover, the HIV-1 vif-minus nonpermissive cell line H9 does not show a similar phenotype for vif-negative HIV-2. We also report a limited effect of APOBEC3G in a single-cycle infectivity assay, where only conserved domains between HIV-1 and HIV-2 Vif proteins influence viral infectivity. Taken together, these results allow us to speculate that viral inhibition by APOBEC3G is not the sole and most important determinant of antiviral activity against HIV-2.     

Subtle mutations in the cysteine region of HIV-1 Vif drastically alter the viral replication phenotype

Mutations were introduced into the region encoding the two cysteine and nearby amino acid residues of human immunodeficiency virus type 1 (HIV-1) Vif protein and, 12 single-amino-acid viral mutants were constructed. Determination of their growth characteristics in two lymphocytic cell lines revealed that only a single amino acid change in the cysteine region greatly altered the replication phenotype. In particular, the four mutants of amino acid 132 of Vif were grouped into three categories on the basis of their growth potentials. These results indicate that the cysteine region of Vif is critical for the cell-dependent replication efficiency of HIV-1.     

Expression of HIV-1 accessory protein Vif is controlled uniquely to be low and optimal by proteasome degradation

While the Vif protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in non-permissive cells, it is rapidly degraded intracellularly. We have previously suggested that the rapid turn-over of Vif is biologically meaningful to prevent detrimental effects of this protein at high expression levels. We now studied the mechanism of Vif degradation by examining the blocking effect of protease inhibitors in pulse/chase experiments and by monitoring the extent of Vif ubiquitination. The rapid turn-over of Vif could be blocked by proteasome inhibitors, and Vif was highly ubiquitinated. Cytoskeletal Vif was found to be more stable than soluble cytosolic Vif. These degradation characteristics of Vif were cell type-independent and observed in both non-permissive and permissive cells. Characterization of a series of vif deletion mutants showed that amino acids predicted to be important for formation of beta-strand structures (amino acid nos. 63-70 and 86-89) were critical for maintaining a normal expression level of Vif and for viral infectivity. Finally, we performed comparative stability analysis of the four HIV-1 accessory proteins. Vif was unique in its short half-life and in the magnitude of the degradation. Taken together, we conclude that the proteasome degradation of HIV-1 Vif is a virologically important process and crucial for the function of Vif.     

Cysteine residues in the Vif protein of human immunodeficiency virus type 1 are essential for viral infectivity.

The infectivity factor of human immunodeficiency virus type 1 (HIV-1), Vif, contains two cysteine residues which are highly conserved among animal lentiviruses. We introduced substitutions of leucine for cysteine residues in the vif gene of a full-length HIV-1 clone to analyze their roles in viral infection. Mutant viruses containing substitutions in either Cys-114, Cys-133, or both displayed a vif-negative infection phenotype similar to that of an isogeneic vif deletion mutant, namely, a cell-dependent complete to partial loss of infectivity. The vif defect could be complemented by cotransfection of mutant viral DNA with a Vif expression vector, and there was no evidence that recombination contributed to the repair of the vif deficiency. The viral protein profile, as determined by immunoblotting, in cells infected with cysteine substitution mutants and that in wild-type virus were similar, including the presence of the 23-kDa Vif polypeptide. In addition, immunoblotting with an antiserum directed against the carboxyl terminus of gp41 revealed that gp41 was intact in cells infected with either wild-type or vif mutant HIV-1, excluding that Vif cleaves the C terminus of gp41. Our results indicate that the cysteines in HIV-1 Vif are critical for Vif function in viral infectivity.     

Identification of HIV-1 Vif Regions Required for CBF-β Interaction and APOBEC3 Suppression

Human immunodeficiency virus type 1 (HIV-1) Vif requires core binding factor β (CBF-β) to degrade the host APOBEC3 restriction factors. Although a minimum domain and certain amino acids of HIV-1 Vif, including hydrophobic residues at the N-terminal, have been identified as critical sites for binding with CBF-β, other regions that potentially mediate this interaction need to be further investigated. Here, we mapped two new regions of HIV-1 Vif that are required for interaction with CBF-β by generating a series of single-site or multiple-site Vif mutants and testing their effect on the suppression of APOBEC3G (A3G) and APOBEC3F (A3F). A number of the mutants, including G84A/SIEW86-89AAAA (84/86–89), E88A/W89A (88/89), G84A, W89A, L106S and I107S in the ⁸⁴GxSIEW⁸⁹ and L¹⁰²ADQLI¹⁰⁷ regions, affected Vif function by disrupting CBF-β binding. These Vif mutants also had altered interactions with CUL5, since CBF-β is known to facilitate the binding of Vif to CUL5. We further showed that this effect was not due to misfolding or conformational changes in Vif, as the mutants still maintained their interactions with other factors such as ElonginB, A3G and A3F. Notably, G84D and D104A had stronger effects on the Vif-CUL5 interaction than on the Vif-CBF-β interaction, indicating that they mainly influenced the CUL5 interaction and implying that the interaction of Vif with CUL5 contributes to the binding of Vif to CBF-β. These new binding interfaces with CBF-β in HIV-1 Vif provide novel targets for the development of HIV-1 inhibitors.     

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.     

Mutational analysis of the human immunodeficiency virus type 1 Vif protein

Lentivirus Vif proteins are potent regulators of virus infectivity. However, relatively little is known about the functional domains, peptide motifs, or residues of any Vif protein. In this report, we present the first extensive mutagenesis analysis of the 192-amino-acid human immunodeficiency virus type 1 (HIV-1) Vif protein. A large number of scanning missense (mostly alanine substitution) and deletion mutations were introduced into the HIV-1HXB3 vif gene, and the resulting proteins were evaluated for the induction of virus infectivity as well as subcellular localization. The results show that amino acids dispersed throughout Vif's linear sequence are important for function. However, because many of the inactive proteins also appear to be mislocalized, we suggest that many of them may actually be misfolded rather lacking an intracellular targeting signal. Interestingly, disruptions within an internal region spanning residues 114 to 146 give rise to mutant proteins that either retain function or are inactive but are not substantially mislocalized. We therefore speculate that this region, which harbors two essential cysteine residues and one essential serine residue, may contain aspects of a putative Vif effector domain.     

Identification of two distinct human immunodeficiency virus type 1 Vif determinants critical for interactions with human APOBEC3G and APOBEC3F

Human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) inhibit replication of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif overcomes these host restriction factors by binding to them and inducing their proteasomal degradation. The Vif-A3G and Vif-A3F interactions are attractive targets for antiviral drug development because inhibiting the interactions could allow the host defense mechanism to control HIV-1 replication. It was recently reported that the Vif amino acids D(14)RMR(17) are important for functional interaction and degradation of the previously identified Vif-resistant mutant of A3G (D128K-A3G). However, the Vif determinants important for functional interaction with A3G and A3F have not been fully characterized. To identify these determinants, we performed an extensive mutational analysis of HIV-1 Vif. Our analysis revealed two distinct Vif determinants, amino acids Y(40)RHHY(44) and D(14)RMR(17), which are essential for binding to A3G and A3F, respectively. Interestingly, mutation of the A3G-binding region increased Vif's ability to suppress A3F. Vif binding to D128K-A3G was also dependent on the Y(40)RHHY(44) region but not the D(14)RMR(17) region. Consistent with previous observations, subsequent neutralization of the D128K-A3G antiviral activity required substitution of Vif determinant D(14)RMR(17) with SEMQ, similar to the SERQ amino acids in simian immunodeficiency virus SIV(AGM) Vif, which is capable of neutralizing D128K-A3G. These studies are the first to clearly identify two distinct regions of Vif that are critical for independent interactions with A3G and A3F. Pharmacological interference with the Vif-A3G or Vif-A3F interactions could result in potent inhibition of HIV-1 replication by the APOBEC3 proteins.     

The regulation of primate immunodeficiency virus infectivity by Vif is cell species restricted: a role for Vif in determining virus host range and cross-species transmission.

The primate immunodeficiency virus Vif proteins are essential for replication in appropriate cultured cell systems and, presumably, for the establishment of productive infections in vivo. We describe experiments that define patterns of complementation between human and simian immunodeficiency virus (HIV and SIV) Vif proteins and address the determinants that underlie functional specificity. Using human cells as virus producers, it was found that the HIV-1 Vif protein could modulate the infectivity of HIV-1 itself, HIV-2 and SIV isolated from African green monkeys (SIVAGM). In contrast, the Vif proteins of SIVAGM and SIV isolated from Sykes' monkeys (SIVSYK) were inactive for all HIV and SIV substrates in human cells even though, at least for the SIVAGM protein, robust activity could be demonstrated in cognate African green monkey cells. These observations suggest that species-specific interactions between Vif and virus-producing cells, as opposed to between Vif and virus components, may govern the functional consequences of Vif expression in terms of inducing virion infectivity. The finding that the replication of murine leukemia virus could also be stimulated by HIV-1 Vif expression in human cells further supported this notion. We speculate that species restrictions to Vif function may have contributed to primate immunodeficiency virus zoonosis.     

Distinct Domains within APOBEC3G and APOBEC3F Interact with Separate Regions of Human Immunodeficiency Virus Type 1 Vif

Human APOBEC3G (A3G) and APOBEC3F (A3F) inhibit the replication of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif overcomes these host restriction factors by binding to them and inducing their degradation. Thus, the Vif-A3G and Vif-A3F interactions are attractive targets for antiviral drug development, as inhibiting these interactions could allow the host defense mechanism to control HIV-1 replication. Recently, it has been reported that amino acids 105 to 156 of A3G are involved in the interaction with Vif; however, to date, the region of A3F involved in Vif binding has not been identified. Using our previously reported Vif mutants that are capable of binding to only A3G (3G binder) or only A3F (3F binder), in conjunction with a series of A3G-A3F chimeras, we have now mapped the APOBEC3-Vif interaction domains. We found that the A3G domain that interacts with the Vif YRHHY region is located between amino acids 126 and 132 of A3G, which is consistent with the conclusions reported in previous studies. The A3F domain that interacts with the Vif DRMR region did not occur in the homologous domain but instead was located between amino acids 283 and 300 of A3F. These studies are the first to identify the A3F domain that interacts with the Vif DRMR region and show that distinct domains of A3G and A3F interact with different Vif regions. Pharmacological inhibition of either or both of these Vif-A3 interactions should prevent the degradation of the APOBEC3 proteins and could be used as a therapy against HIV-1.     

The HIV-1 Vif protein mediates degradation of Vpr and reduces Vpr-induced cell cycle arrest

Prior work has implicated viral protein R (Vpr) in the arrest of human immunodeficiency virus type 1 (HIV-1)-infected cells in the G2 phase of the cell cycle, associated with increased viral replication and host cell apoptosis. We and others have recently shown that virion infectivity factor (Vif ) also plays a role in the G2 arrest of HIV-1-infected cells. Here, we demonstrate that, paradoxically, at early time points postinfection, Vif expression blocks Vpr-mediated G2 arrest, while deletion of Vif from the HIV-1 genome leads to a marked increase in G2 arrest of infected CD4 T-cells. Consistent with this increased G2 arrest, T-cells infected with Vif-deleted HIV-1 express higher levels of Vpr protein than cells infected with wild-type virus. Further, expression of exogenous Vif inhibits the expression of Vpr, associated with a decrease in G2 arrest of both infected and transfected cells. Treatment with the proteasome inhibitor MG132 increases Vpr protein expression and G2 arrest in wild-type, but not Vif-deleted, NL4-3-infected cells, and in cells cotransfected with Vif and Vpr. In addition, Vpr coimmunoprecipitates with Vif in cotransfected cells in the presence of MG132. This suggests that inhibition of Vpr by Vif is mediated at least in part by proteasomal degradation, similar to Vif-induced degradation of APOBEC3G. Together, these data show that Vif mediates the degradation of Vpr and modulates Vpr-induced G2 arrest in HIV-1-infected T-cells.     

Vif substitution enables persistent infection of pig-tailed macaques by human immunodeficiency virus type 1

Among Old World monkeys, pig-tailed macaques (Pt) are uniquely susceptible to human immunodeficiency virus type 1 (HIV-1), although the infection does not persist. We demonstrate that the susceptibility of Pt T cells to HIV-1 infection is due to the absence of postentry inhibition by a TRIM5 isoform. Notably, substitution of the viral infectivity factor protein, Vif, with that from pathogenic SIVmne enabled replication of HIV-1 in Pt T cells in vitro. When inoculated into juvenile pig-tailed macaques, the Pt-tropic HIV-1 persistently replicated for more than 1.5 to 2 years, producing low but measurable plasma viral loads and persistent proviral DNA in peripheral blood mononuclear cells. It also elicited strong antibody responses. However, there was no decline in CD4(+) T cells or evidence of disease. Surprisingly, the Pt-tropic HIV-1 was rapidly controlled when inoculated into newborn Pt macaques, although it transiently rebounded after 6 months. We identified two notable differences between the Pt-tropic HIV-1 and SIVmne. First, SIV Vif does not associate with Pt-tropic HIV-1 viral particles. Second, while Pt-tropic HIV-1 degrades both Pt APOBEC3G and APOBEC3F, it prevents their inclusion in virions to a lesser extent than pathogenic SIVmne. Thus, while SIV Vif is necessary for persistent infection by Pt-tropic HIV-1, improved expression and inhibition of APOBEC3 proteins may be required for robust viral replication in vivo. Additional adaptation of the virus may also be necessary to enhance viral replication. Nevertheless, our data suggest the potential for the pig-tailed macaque to be developed as an animal model of HIV-1 infection and disease.     

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.