Vif and the p55(Gag) polyprotein of human immunodeficiency virus type 1 are present in colocalizing membrane-free cytoplasmic complexes.
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The Vif protein of human immunodeficiency virus type 1 (HIV-1) is a potent regulator of viral infectivity. Current data posit that Vif functions late in replication to modulate assembly, budding, and/or maturation. Consistent with this model, earlier indirect immunofluorescence analyses of HIV-1-infected cells demonstrated that Vif and Gag colocalize to a substantial degree (J. H. M. Simon, R. A. M. Fouchier, T. E. Southerling, C. B. Guerra, C. K. Grant, and M. H. Malim, J. Virol. 71:5259-5267, 1997). Here, we describe a series of subcellular fractionation studies which indicate that Vif and the p55(Gag) polyprotein are present in membrane-free cytoplasmic complexes that copurify in sucrose density gradients and are stable in nonionic detergents. Both Vif and Gag are targeted to these complexes independent of each other, and their association with them appears to be mediated by protein-protein interactions. We propose that these complexes may represent viral assembly intermediates and that Vif is appropriately localized to influence the final stages of the viral life cycle and, therefore, the infectivity of progeny virions. (+info)
Mutational analysis of the human immunodeficiency virus type 1 Vif protein.
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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. (+info)
The feline immunodeficiency virus vif gene is required for productive infection of feline peripheral blood mononuclear cells and monocyte-derived macrophages.
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The role of the feline immunodeficiency virus (FIV) vif gene in establishing productive infection in feline peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) was examined in cell culture systems. A 375-bp deletion was introduced into the vif gene of the wild-type FIV-pPPR infectious molecular clone to produce Vif deletion mutant FIV-pPPRDeltavif. This mutant FIV proviral construct expressed FIV proteins p24gag and gp100env in transfected Crandell feline kidney cells as measured by immunoprecipitation and Western blot analysis as well as immunocytochemical analysis; these cultures produced very low levels of virus by cocultivation of transfected cells with PBMCs and K-258 cells, as measured by production of p24gag. Replication kinetics of wild-type and vif-deleted virus were compared in PBMCs and monocyte-derived macrophages (MDMs) by infection with cell-free virus preparations. Similar to findings with other lentiviruses, the vif gene was found to be essential for establishment of productive FIV infection in both PBMCs and MDMs. This study indicates that vif is essential for productive FIV infection of host target cells in vitro and that FIV-pPPRDeltavif may be an excellent candidate viral mutant for attenuated virus vaccine studies. (+info)
Pseudotyping human immunodeficiency virus type 1 by vesicular stomatitis virus G protein does not reduce the cell-dependent requirement of vif for optimal infectivity: functional difference between Vif and Nef.
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The functions of Vif and Nef in human immunodeficiency virus type 1 (HIV-1) infection have some similarities: Vif- and Nef-dependent enhancement of HIV-1 replication is cell type-specific, and defective mutations in these genes result in restricted proviral DNA synthesis in infected cells. It has recently been shown that pseudotyping HIV-1 by the envelope glycoprotein of vesicular stomatitis virus (VSV-G) targets HIV-1 entry to an endocytic pathway and suppresses the requirement of Nef for virus infectivity. In this study, we examined whether VSV-G pseudotyping suppresses the requirement of Vif for HIV-1 infectivity. It was found that pseudotyping HIV-1 by VSV-G did not compensate for the Vif function. Together with the findings that Vif does not influence virus binding/entry and virion incorporation of Env, it is concluded that Vif enhances HIV-1 infectivity at the post-entry step(s) independently of the Env function by a different mechanism to that of Nef. (+info)
Feline immunodeficiency virus Vif localizes to the nucleus.
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Monoclonal antibodies prepared against recombinant Vif derived from the 34TF10 strain of feline immunodeficiency virus (FIV) were used to assess the expression and localization of Vif in virus-infected cells. Analyses by Western blotting and by immunoprecipitation from cells infected with FIV-34TF10 revealed the presence of a single 29-kDa species specific for virus-infected cells. Confirmation of antibody specificity was also performed by specific immunoprecipitation of in vitro-transcribed and -translated recombinant Vif. Localization experiments were also performed on virus-infected cells, using different fixation procedures. Results for methanol fixation protocols similar to those reported for localization of human immunodeficiency virus (HIV) Vif showed a predominant cytoplasmic localization for FIV Vif, very similar to localization of HIV type 1 Vif and virtually identical to the localization observed for the Gag antigens of the virus. However, with milder fixation procedures that used 2% formaldehyde at 4 degrees C, FIV Vif was strongly evident in the nucleus. The localization was distinct from the nuclear localization noted with Rev and did not involve the nucleolus. Attempts to show colocalization or coprecipitation of Vif with Gag antigens were unsuccessful. In addition, Vif was not detected in purified FIV virions. The results are consistent with the notion that the primary role of Vif in virus infection initiates in the nucleus. (+info)
Partial rescue of the Vif-negative phenotype of mutant human immunodeficiency virus type 1 strains from nonpermissive cells by intravirion reverse transcription.
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Virion infectivity factor (Vif) is a protein encoded by human immunodeficiency virus type I (HIV-1) and is essential for viral replication. It appears that Vif functions in the virus-producing cells and affects viral assembly. Viruses with defects in the vif gene (vif-) generated from the "nonpermissive cells" are not able to complete reverse transcription. In previous studies, it was demonstrated that defects in the vif gene also affect endogenous reverse transcription (ERT) when mild detergents were utilized to permeabilize the viral envelope. In this report, we demonstrate that defects in the vif gene have much less of an effect on ERT if detergent is not used. When ERT was driven by addition of deoxyribonucleoside triphosphates (dNTPs) at high concentrations, certain levels of plus-strand viral DNA could also be achieved. Interestingly, if vif- viruses, generated from nonpermissive cells and harboring large quantities of viral DNA generated by ERT, were allowed to infect permissive cells, they could partially bypass the block at intracellular reverse transcription, through which vif- viruses without dNTP treatment could not pass. Consequently, viral infectivity can be partially rescued from the vif- phenotype. Based on our observations, we suggest that vif defects may cause the reverse transcription complex (RT complex) to become sensitive to mild detergent treatments within HIV-1 virions and become unstable in the target cells, such that the process of reverse transcription cannot be efficiently supported. Further dissection of RT complexes of vif- viruses may be key to uncovering the molecular mechanism(s) of Vif in HIV-1 pathogenesis. (+info)
Codon-substitution models for heterogeneous selection pressure at amino acid sites.
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Comparison of relative fixation rates of synonymous (silent) and nonsynonymous (amino acid-altering) mutations provides a means for understanding the mechanisms of molecular sequence evolution. The nonsynonymous/synonymous rate ratio (omega = d(N)d(S)) is an important indicator of selective pressure at the protein level, with omega = 1 meaning neutral mutations, omega < 1 purifying selection, and omega > 1 diversifying positive selection. Amino acid sites in a protein are expected to be under different selective pressures and have different underlying omega ratios. We develop models that account for heterogeneous omega ratios among amino acid sites and apply them to phylogenetic analyses of protein-coding DNA sequences. These models are useful for testing for adaptive molecular evolution and identifying amino acid sites under diversifying selection. Ten data sets of genes from nuclear, mitochondrial, and viral genomes are analyzed to estimate the distributions of omega among sites. In all data sets analyzed, the selective pressure indicated by the omega ratio is found to be highly heterogeneous among sites. Previously unsuspected Darwinian selection is detected in several genes in which the average omega ratio across sites is <1, but in which some sites are clearly under diversifying selection with omega > 1. Genes undergoing positive selection include the beta-globin gene from vertebrates, mitochondrial protein-coding genes from hominoids, the hemagglutinin (HA) gene from human influenza virus A, and HIV-1 env, vif, and pol genes. Tests for the presence of positively selected sites and their subsequent identification appear quite robust to the specific distributional form assumed for omega and can be achieved using any of several models we implement. However, we encountered difficulties in estimating the precise distribution of omega among sites from real data sets. (+info)
Cellular and viral specificities of human immunodeficiency virus type 1 vif protein.
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The vif gene of human immunodeficiency virus type 1 (HIV-1) greatly enhances the infectivity of HIV-1 virions that are released from cells classified as nonpermissive (e.g., lymphocytes, macrophages, and H9 leukemic T cells) but is irrelevant in permissive cells (e.g., HeLa or COS cells). Recently, it was reported that vif expression in nonpermissive cells dramatically increases infectivity not only of HIV-1 but also of other enveloped viruses, including murine leukemia viruses (MLVs). This was surprising in part because MLVs and other murine retroviruses lack vif genes yet replicate efficiently in T lymphocytes. To investigate these issues, we first developed improved methods for producing substantial quantities of HIV-1 virions with vif deletions from healthy H9 cells. These virions had approximately the same amounts of major core proteins and envelope glycoproteins as the control wild-type virions but were only approximately 1% as infectious. We then produced H9 cells that contained wild-type or vif deletion HIV-gpt proviruses, which lack a functional env gene. After superinfection with either xenotropic or amphotropic MLVs, these cells released HIV-gpt virions pseudotyped with an MLV envelope plus replication-competent MLV. Interestingly, the pseudotyped HIV-gpt (vif deletion) virions were noninfectious, whereas the MLV virions simultaneously released from the same H9 cells were fully infectious. These results strongly suggest that the Vif protein functions in a manner that is both cell specific and at least substantially specific for HIV-1 and related lentiviruses. In addition, these results confirm that vif deletion HIV-1 virions from nonpermissive cells are blocked at a postpenetration stage of the infection pathway. (+info)