The viral infectivity factor (Vif) of HIV-1 unveiled

The viral infectivity factor (Vif) of HIV type-1 (HIV-1) is essential for efficient viral replication, yet was, until recently, enigmatic. This resulted from the complexity and cellular specificity of its function and the correspondingly complex systems that are required for its investigation. These limitations have been overcome and Vif function has been rapidly elucidated, with implications for the development of drugs to block its activity. These studies have revealed a novel component of the innate immune system, APOBEC3G, that lethally hypermutates retroviruses, including HIV-1. For HIV-1, the competition between the virus and APOBEC3G is tipped in favor of the invader by Vif, which binds to APOBEC3G and triggers its polyubiquitination and rapid degradation, thereby preventing its entry into progeny virions.     

Small-molecule inhibition of HIV-1 Vif

The HIV-1 protein Vif, essential for in vivo viral replication, targets the human DNA-editing enzyme, APOBEC3G (A3G), which inhibits replication of retroviruses and hepatitis B virus. As Vif has no known cellular homologs, it is an attractive, yet unrealized, target for antiviral intervention. Although zinc chelation inhibits Vif and enhances viral sensitivity to A3G, this effect is unrelated to the interaction of Vif with A3G. We identify a small molecule, RN-18, that antagonizes Vif function and inhibits HIV-1 replication only in the presence of A3G. RN-18 increases cellular A3G levels in a Vif-dependent manner and increases A3G incorporation into virions without inhibiting general proteasome-mediated protein degradation. RN-18 enhances Vif degradation only in the presence of A3G, reduces viral infectivity by increasing A3G incorporation into virions and enhances cytidine deamination of the viral genome. These results demonstrate that the HIV-1 Vif-A3G axis is a valid target for developing small molecule-based new therapies for HIV infection or for enhancing innate immunity against viruses.     

Unique characteristics of HIV-1 Vif expression

We examined the steady-state expression in cells of four accessory proteins of human immunodeficiency virus type 1 (HIV-1). For this purpose, a series of single gene expression vectors for these viral proteins were constructed and were monitored for their production by transfection. Among them, the expression level of Vif was found to be lowest in both the absence and presence of APOBEC3G. In addition, we noticed the presence of its truncated form, which was not observed for the other accessory proteins. When a subgenomic vector was used for transfection, authentic and several small forms of Vif were produced. By mutational analysis, these forms were demonstrated to be mutant Vif proteins translated from M8, M16 and M29. When a full-length molecular clone was used, the smaller versions of Vif were hardly observed. Functional analysis of these mutant Vif proteins showed that they are incapable of modulating viral infectivity. The results described above, i.e. the low steady-state expression and the presence of truncated forms, represent the unique characteristics of HIV-1 Vif.     

Status of APOBEC3G/F in cells and progeny virions modulated by Vif determines HIV-1 infectivity

We examined various HIV-1 Vif mutants for interaction with APOBEC3 proteins (A3G/A3F). All replication-defective proviral mutants were found to carry A3G/A3F in virions, and of these, a replication-defective mutant with Vif that binds to A3G in cells but not in virions was noted. Furthermore, a mutant Vif protein that suppresses A3F activity but does not exclude A3F from virions was identified. We also showed that incorporation of Vif into virions is dependent on its interaction with A3G/A3F. Taken together, we concluded that functional binding of Vif to A3G/A3F in cells and/or virions is critical for viral infectivity.     

Potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type 1 (HIV-1) Vif proteins

Virion infectivity factor (Vif) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) in vivo, but its function remains uncertain. Recently, we have shown that Vif proteins are able to form multimers, including dimers, trimers, or tetramers. Because the multimerization of Vif proteins is required for Vif function in the viral life cycle, we propose that it could be a novel target for anti-HIV-1 therapeutics. Through a phage peptide display method, we have identified a set of 12-mer peptides containing a PXP motif that binds to HIV-1 Vif protein. These proline-enriched peptides potently inhibited the Vif-Vif interaction in vitro. We have also screened a set of synthesized Vif peptides (15-mer), which covers all the amino acids of the HIV-1 Vif protein sequence, for their ability to inhibit the Vif-Vif interaction in vitro. We demonstrated that Vif-derived proline-enriched peptides that contain the (161)PPLP(164) domain are able to inhibit the Vif-Vif interaction. Conversely, the deletion of the (161)PPLP(164) domain of Vif protein will significantly impair the capability of Vif proteins to interact with each other, indicating that the (161)PPLP(164) domain plays a key role in Vif multimerization. All these results demonstrate that the proline-enriched peptides block the multimerization of Vif through interfering with the polyproline interfaces of Vif formed by (161)PPLP(164) domain. Moreover, these peptides which inhibit the Vif-Vif interaction in vitro potently inhibit HIV-1 replication in the "nonpermissive" T-cells. We propose that this study starts a novel strategy to develop structural diverse inhibitors of Vif such as peptidomimetics or small organic molecules.     

Optimization of HIV-1 infectivity assays

HIV-1 reporter cell lines are the backbone of diagnostic assays, vaccine and drug development efforts. Performing HIV-1 infection experiments in a T cell background is desirable for many reasons. However, a low susceptibility to infection with primary patient isolates in available reporter T cell lines has limited such efforts. We here demonstrate that optimization of HIV-1 receptor expression and the utilization of serum free medium compositions can increase susceptibility of reporter T cell lines to HIV-1 infection by up to two orders of magnitude.     

The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G

We have obtained the 1.7 Å crystal structure of FIV protease (PR) in which 12 critical residues around the active site have been substituted with the structurally equivalent residues of HIV PR (12X FIV PR). The chimeric PR was crystallized in complex with the broad-based inhibitor TL-3, which inhibits wild type FIV and HIV PRs, as well as 12X FIV PR and several drug-resistant HIV mutants [1–4]. Biochemical analyses have demonstrated that TL-3 inhibits these PRs in the order HIV PR > 12X FIV PR > FIV PR, with K_i values of 1.5 nM, 10 nM, and 41 nM, respectively [2–4]. Comparison of the crystal structures of the TL-3 complexes of 12X FIV and wild-typeFIV PR revealed theformation of additinal van der Waals interactions between the enzyme inhibitor in the mutant PR. The 12X FIV PR retained the hydrogen bonding interactions between residues in the flap regions and active site involving the enzyme and the TL-3 inhibitor in comparison to both FIV PR and HIV PR. However, the flap regions of the 12X FIV PR more closely resemble those of HIV PR, having gained several stabilizing intra-flap interactions not present in wild type FIV PR. These findings offer a structural explanation for the observed inhibitor/substrate binding properties of the chimeric PR.     

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.     

APOBEC3F Determinants of HIV-1 Vif Sensitivity

HIV-1 Vif counteracts restrictive APOBEC3 proteins by targeting them for proteasomal degradation. To determine the regions mediating sensitivity to Vif, we compared human APOBEC3F, which is HIV-1 Vif sensitive, with rhesus APOBEC3F, which is HIV-1 Vif resistant. Rhesus-human APOBEC3F chimeras and amino acid substitution mutants were tested for sensitivity to HIV-1 Vif. This approach identified the α3 and α4 helices of human APOBEC3F as important determinants of the interaction with HIV-1 Vif.     

Enhanced infectivity of HIV-1 by X4 HIV-1 coinfection

Two strains of human immunodeficiency virus type 1 (HIV-1) expressing different reporters, human placental alkaline phosphatase (PLAP) and murine heat stable antigen (HSA, CD24), were used for dual infection. Flow cytometric analysis enabled us to distinguish cells not only infected with individual reporter virus but also superinfected with both reporter viruses. When the CD4 positive T cell line, PM1, was dually infected by both reporter viruses with different coreceptor utilization, coinfection with CXCR4-tropic HIV-1 (X4 HIV-1) expressing one reporter increased the rate of cells infected with HIV-1 expressing another reporter. This enhancement was accompanied by an increased level of p24 antigen Gag in culture supernatant, indicating that infectivity of HIV-1 was augmented by X4 HIV-1 coinfection. The CXCR4 antagonist, T140 eliminated this enhancement, suggesting the role of X4 envelope via CXCR4. These results imply the role of X4 HIV-1 at the late stage of infection.     

Mass spectrometry analysis of HIV-1 Vif reveals an increase in ordered structure upon oligomerization in regions necessary for viral infectivity

HIV-1 Vif, an accessory protein in the viral genome, performs an important role in viral pathogenesis by facilitating the degradation of APOBEC3G, an endogenous cellular inhibitor of HIV-1 replication. In this study, intrinsically disordered regions are predicted in HIV-1 Vif using sequence-based algorithms. Intrinsic disorder may explain why traditional structure determination of HIV-1 Vif has been elusive, making structure-based drug design impossible. To characterize HIV-1 Vif's structural topology and to map the domains involved in oligomerization we used chemical cross-linking, proteolysis, and mass spectrometry. Cross-linking showed evidence of monomer, dimer, and trimer species via denaturing gel analysis and an additional tetramer via western blot analysis. We identified 47 unique linear peptides and 24 (13 intramolecular; 11 intermolecular) noncontiguous, cross-linked peptides, among the noncross-linked monomer, cross-linked monomer, cross-linked dimer, and cross-linked trimer samples. Almost complete peptide coverage of the N-terminus is observed in all samples analyzed, however reduced peptide coverage in the C-terminal region is observed in the dimer and trimer samples. These differences in peptide coverage or "protections" between dimer and trimer indicate specific differences in packing between the two oligomeric forms. Intramolecular cross-links within the monomer suggest that the N-terminus is likely folded into a compact domain, while the C-terminus remains intrinsically disordered. Upon oligomerization, as evidenced by the intermolecular cross-links, the C-terminus of one Vif protein becomes ordered by wrapping back on the N-terminal domain of another. In addition, the majority of the intramolecular cross-links map to regions that have been previously reported to be necessary for viral infectivity. Thus, this data suggests HIV-1 Vif is in a dynamic equilibrium between the various oligomers potentially allowing it to interact with other binding partners.     

HIV-1辅助蛋白Vif的结构与功能

HIV-1 Vif(viral infectivity factor)蛋白是由保守的vif基因编码的碱性蛋白质,是HIV-1病毒的辅助调节蛋白之一.研究表明Vif蛋白具有调节病毒侵入、组装、出芽和成熟等功能.此外,Vif蛋白能够特异性地与体内抗病毒因子APOBEC3G相互作用,增强病毒的感染性.因此,针对HIV-1Vif蛋白进行抑制剂设计已经成为抗HIV药物研究的热点之一.本文对HIV-1Vif蛋白的结构与功能研究的最新进展进行了综述.     

Cyclophilin A regulates HIV-1 infectivity, as demonstrated by gene targeting in human T cells

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein binds most members of the cyclophilin family of peptidyl-prolyl isomerases. Of 15 known human cyclophilins, cyclophilin A (CypA) has been the focus of investigation because it was detected in HIV-1 virions. To determine whether CypA promotes HIV-1 replication, we deleted the gene encoding CypA (PPIA) in human CD4(+) T cells by homologous recombination. HIV-1 replication in PPIA(-/-) cells was decreased and not inhibited further by cyclosporin or gag mutations that disrupt Gag's interaction with cyclophilins, indicating that no other cyclophilin family members promote HIV-1 replication. The defective replication phenotype was specific for wild-type HIV-1 since HIV-2/SIV isolates, as well as HIV-1 bearing a gag mutation that confers cyclosporin resistance, replicated the same in PPIA(+/+) and PPIA(-/-) cells. Stable re-expression of CypA in PPIA(-/-) cells restored HIV-1 replication to an extent that correlated with steady-state levels of CypA. Finally, virions from PPIA(-/-) cells possessed no obvious biochemical abnormalities but were less infectious than virions from wild-type cells. These data formally demonstrate that CypA regulates the infectivity of HIV-1 virions.