HIV-1 Viral Infectivity Factor (Vif) Alters Processive Single-stranded DNA Scanning of the Retroviral Restriction Factor APOBEC3G

APOBEC3G is a retroviral restriction factor that can inhibit the replication of human immunodeficiency virus, type 1 (HIV-1) in the absence of the viral infectivity factor (Vif) protein. Virion-encapsidated APOBEC3G can deaminate cytosine to uracil in viral (−)DNA, which leads to hypermutation and inactivation of the provirus. APOBEC3G catalyzes these deaminations processively on single-stranded DNA using sliding and jumping movements. Vif is thought to primarily overcome APOBEC3G through an interaction that mediates APOBEC3G ubiquitination and results in its proteasomal degradation. However, Vif may also inhibit APOBEC3G mRNA translation, virion encapsidation, and deamination activity. Here we investigated the molecular mechanism of Vif_IIIB- and Vif_HXB2-mediated inhibition of APOBEC3G deamination activity. Biochemical assays using a model HIV-1 replication assay and synthetic single-stranded or partially double-stranded DNA substrates demonstrated that APOBEC3G has an altered processive mechanism in the presence of Vif. Specifically, Vif_HXB2 inhibited the jumping and Vif_IIIB inhibited the sliding movements of APOBEC3G. The absence of such an effect by Vif on degradation-resistant APOBEC3G D128K indicates that a Vif-APOBEC3G interaction mediates this effect. That the partially processive APOBEC3G was less effective at inducing mutagenesis in a model HIV-1 replication assay suggests that Vif co-encapsidation with APOBEC3G can promote sublethal mutagenesis of HIV-1 proviral DNA.     

病毒感染因子在APOBEC3G抗病毒中的拮抗作用

病毒感染因子(virion infectivity factor, Vif)是人免疫缺陷病毒(human im_mu_n_o_de_fi_cien_cy virus, HIV)的6个辅助蛋白之一, 是病毒进行有效复制所必需的.由于Vif功能的复杂性以及对相应复合物体系的不了解, 一直以来, 对Vif的研究进展缓慢.直到2002年发现载脂蛋白B mRNA编辑酶催化多肽样蛋白3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G, APOBEC3G)是存在于细胞内的一种天然抗病毒因子后, Vif的功能才被逐步阐明.APOBEC3G主要通过嘧啶脱氨基活性使HIV-1的负链DNA在逆转录过程中发生致死性超突变, 从而起到抗病毒作用.HIV-1基因编码Vif来拮抗APOBEC3G, 二者在宿主细胞内达到动态平衡.Vif通过介导APOBEC3G降解、减少在胞内的表达、阻碍其向病毒粒子的包装以及促使其装配成无活性的高分子质量复合体等多种途径起到中和作用.对Vif/APOBEC3G相互作用及其调节机制的进一步研究, 将为新型抗HIV-1病毒药物的研制与开发提供理论依据.     

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.     

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.     

Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.     

HIV-1 Vif and APOBEC3G: Multiple roads to one goal

The viral infectivity factor, Vif, of human immunodeficiency virus type 1, HIV-1, has long been shown to promote viral replication in vivo and to serve a critical function for productive infection of non-permissive cells, like peripheral blood mononuclear cells (PBMC). Vif functions to counteract an anti-retroviral cellular factor in non-permissive cells named APOBEC3G. The current mechanism proposed for protection of the virus by HIV-1 Vif is to induce APOBEC3G degradation through a ubiquitination-dependent proteasomal pathway. However, a new study published in Retrovirology by Strebel and colleagues suggests that Vif-induced APOBEC3G destruction may not be required for Vif's virus-protective effect. Strebel and co-workers show that Vif and APOBEC3G can stably co-exist, and yet viruses produced under such conditions are fully infectious. This new result highlights the notion that depletion of APOBEC3G is not the sole protective mechanism of Vif and that additional mechanisms exerted by this protein can be envisioned which counteract APOBEC3G and enhance HIV infectivity.     

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.     

APOBEC3G与Vif在HIV-1感染中的作用

载脂蛋白B mRNA编辑催化多肽样（apolipoprotein B mRNA-editing catalytic polypeptide-like,APOBEC）蛋白是一组胞嘧啶脱氨基酶,具有天然的抗病毒活性,对多种病毒具有抑制作用,特别是逆转录病毒. APOBEC3蛋白能够抑制人类免疫缺陷病毒(HIV-1)的感染,其中APOBEC3G和APOBEC3F的作用最强. APOBEC3G能够通过胞嘧啶脱氨基作用和非胞嘧啶脱氨基作用抑制病毒感染. HIV-1病毒感染因子(Vif) 蛋白主要经泛素-蛋白酶体途径介导APOBEC3G降解,从而拮抗其抗病毒作用. APOBEC3G和Vif之间相互作用的研究对于寻求新的抗HIV治疗靶点具有重要意义.     

Natural Polymorphisms in Human APOBEC3H and HIV-1 Vif Combine in Primary T Lymphocytes to Affect Viral G-to-A Mutation Levels and Infectivity

The Vif protein of HIV-1 allows virus replication by degrading several members of the host-encoded APOBEC3 family of DNA cytosine deaminases. Polymorphisms in both host APOBEC3 genes and the viral vif gene have the potential to impact the extent of virus replication among individuals. The most genetically diverse of the seven human APOBEC3 genes is APOBEC3H with seven known haplotypes. Overexpression studies have shown that a subset of these variants express stable and active proteins, whereas the others encode proteins with a short half-life and little, if any, antiviral activity. We demonstrate that these stable/unstable phenotypes are an intrinsic property of endogenous APOBEC3H proteins in primary CD4+ T lymphocytes and confer differential resistance to HIV-1 infection in a manner that depends on natural variation in the Vif protein of the infecting virus. HIV-1 with a Vif protein hypo-functional for APOBEC3H degradation, yet fully able to counteract APOBEC3D, APOBEC3F, and APOBEC3G, was susceptible to restriction and hypermutation in stable APOBEC3H expressing lymphocytes, but not in unstable APOBEC3H expressing lymphocytes. In contrast, HIV-1 with hyper-functional Vif counteracted stable APOBEC3H proteins as well as all other endogenous APOBEC3s and replicated to high levels. We also found that APOBEC3H protein levels are induced over 10-fold by infection. Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue. These data combine to strongly suggest that stable APOBEC3H haplotypes present as in vivo barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached.     

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.     

Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vif is required for productive infection of T lymphocytes and macrophages. Virions produced in the absence of Vif have abnormal core morphology and those produced in primary T cells carry immature core proteins and low levels of mature capsid (M. Simm, M. Shahabuddin, W. Chao, J. S. Allan, and D. J. Volsky, J. Virol. 69:4582-4586, 1995). To investigate whether Vif influences the activity of HIV-1 protease (PR), the viral enzyme which is responsible for processing Gag and Gag-Pol precursor polyproteins into mature virion components, we transformed bacteria to inducibly express truncated Gag-Pol fusion proteins and Vif. We examined the cleavage of polyproteins consisting of matrix to PR (Gag-PR), capsid to PR (CA-PR), and p6Pol to PR (p6Pol-PR) and evaluated HIV-1 protein processing at specific sites by Western blotting using antibodies against matrix, capsid, and PR proteins. We found that Vif modulates HIV-1 PR activity in bacteria mainly by preventing the release of mature MA and CA from Gag-PR, CA from CA-PR, and p6Pol from p6Pol-PR, with other cleavages being less affected. Using subconstructs of Vif, we mapped this activity to the N-terminal half of the molecule, thus identifying a new functional domain of Vif. Kinetic study of p6Pol-PR autocatalysis in the presence or absence of Vif revealed that Vif and N'Vif reduce the rate of PR-mediated proteolysis of this substrate. In an assay of in vitro proteolysis of a synthetic peptide substrate by purified recombinant PR we found that recombinant Vif and the N-terminal half of the molecule specifically inhibit PR activity at a molar ratio of the N-terminal half of Vif to PR of about 1. These results suggest a mechanism and site of action of Vif in HIV-1 replication and demonstrate novel regulation of a lentivirus PR by an autologous viral protein acting in trans.     

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.     

Inhibition of human immunodeficiency virus type 1 (HIV-1) replication by a two-amino-acid insertion in HIV-1 Vif from a nonprogressing mother and child

We studied a 15-year-old girl, patient X, who has maintained consistently low plasma loads of human immunodeficiency virus type 1 (HIV-1) RNA, as well as normal and stable CD4(+) T-cell concentrations. She has presented no clinical manifestations of AIDS, despite having only received zidovudine monotherapy for a part of her life. Patient X's HIV-positive mother (patient Y) has also not progressed to AIDS and has never been treated with antiretroviral agents. HIV-1 isolated from patient X replicated poorly in human peripheral blood mononuclear cells (PBMC). In order to map the determinant of the poor growth of patient X's isolate, viral sequences from patient X were determined and examined for insertion or deletion mutations. These sequences contained a two-amino-acid insertion mutation in the Vif gene, which was also observed in uncultured PBMC acquired at different times. Furthermore, Vif sequences harbored by patient Y contained the identical mutation. These observations suggest that polymorphic HIV-1 was transmitted to patient X perinatally 15 years previously and has been maintained since that time. Recombinant HIV-1, engineered with Vif sequences from patient X, replicated in PBMC to levels approximately 20-fold lower than that of wild type. Removal of the insertion mutation from this recombinant restored replication efficiency to wild-type levels, while introduction of the insertion mutation into wild-type Vif sequences resulted in greatly decreased replication. Furthermore, Vif protein from patient X's HIV-1 was aberrantly cleaved, suggesting a mechanism for loss of Vif function. Since HIV-1 containing these sequences replicates poorly, the implication is that the two-amino-acid insertion mutation in Vif contributes significantly to the nonprogressor status of this mother and child. Further studies of these sequences might provide information regarding contributions of Vif structure and/or function to HIV-1 virulence.