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1.
J Virol ; 78(19): 10574-81, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15367624

ABSTRACT

Virion infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary human CD4 T lymphocytes and macrophages. Vif overcomes the HIV-inhibitory effects of cellular factor APOBEC3G, which has cytidine deaminase activity. We previously reported the isolation of a Vif-interacting ring finger protein, Triad 3, from a human leukocyte cDNA library, using the yeast two-hybrid system. The full-length cellular protein homologue of Triad 3 has been recently identified as the zinc finger protein inhibiting NF-kappaB (ZIN). Sequence analysis indicates that Triad 3 protein contains all four major ring-like motifs of ZIN. We report here that ZIN binds to purified Vif in vitro and that Triad 3/ZIN interacts with HIV-1 Vif in transfected human 293T cells, as demonstrated by coimmunoprecipitation. To test the biological relevance of this interaction, we produced infectious HIV-1 NL4.3 in the presence or absence of cotransfected ZIN. HIV-1 NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were twofold less infectious in a single-cycle infectivity assay than virus produced in the absence of exogenous ZIN. It was further shown that cells infected with HIV NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were impaired in viral DNA synthesis by twofold. The impairment in viral reverse transcription and the reduction in single-cycle viral infectivity were both shown to be dependent on the presence of Vif in the virus producer cells. The possible mechanisms by which ZIN interferes with the early events of HIV-1 replication are discussed.


Subject(s)
Carrier Proteins/metabolism , Gene Products, vif/metabolism , HIV-1/pathogenicity , Intracellular Signaling Peptides and Proteins , Amino Acid Sequence , Blotting, Western , Carrier Proteins/genetics , Cell Line , DNA, Viral/analysis , DNA, Viral/biosynthesis , Gene Deletion , Gene Expression Regulation , Gene Products, vif/genetics , Gene Products, vif/isolation & purification , HIV-1/genetics , Humans , Molecular Sequence Data , Precipitin Tests , Protein Binding , RNA, Messenger/analysis , Transcription, Genetic , Ubiquitin-Protein Ligases , Virus Replication/physiology , Zinc Fingers , vif Gene Products, Human Immunodeficiency Virus
2.
J Virol ; 73(2): 1460-7, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9882352

ABSTRACT

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary blood-derived lymphocytes, macrophages, and certain human T-cell lines. It has been shown that Vif is associated with HIV-1 virions purified by sucrose density-equilibrium gradient analysis. However, the specificity of Vif incorporation into virions has not been determined. Moreover, recent studies have demonstrated that standard HIV-1 particle preparations created with sucrose density-equilibrium gradients are contaminated with cell-derived microvesicles. Here we demonstrate, as previously reported, that Vif cosediments with HIV-1 particles in sucrose density-equilibrium gradient analysis. However, we also found that, when Vif was expressed in the absence of all other HIV-1-encoded gene products and then isolated by sucrose density-equilibrium gradient centrifugation from extracellular supernatants, its sedimentation pattern was largely unaltered, suggesting that Vif can be secreted from cells. Using a newly developed OptiPrep velocity gradient method, we were able to physically separate most of the extracellular Vif from the HIV-1 virions without disrupting the infectivity of the virus. By titrating serial dilutions of purified Vif and Gag against the viral peak fraction in the OptiPrep gradient, we demonstrate that <1.0 Vif molecule per virion was present. This study shows that Vif is not significantly present in HIV-1 virions, a finding which is consistent with the idea that Vif functions predominantly in the virus-producing cells during virus assembly. The OptiPrep velocity gradient technique described here could be an easy and rapid way to purify HIV and other enveloped viruses from microvesicles and/or cell debris.


Subject(s)
Gene Products, vif/isolation & purification , HIV-1 , Animals , COS Cells , Centrifugation, Density Gradient , Humans , Virion , vif Gene Products, Human Immunodeficiency Virus
4.
J Virol ; 70(9): 6106-11, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8709234

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) Vif protein is necessary at the time of viral particle formation yet functionally manifests its effect after virions enter target cells. This suggests that Vif either acts on another viral protein or is itself incorporated into particles. In this study, we have examined the latter possibility. We confirm our previous observation that Vif is incorporated into human immunodeficiency virus type 1 virions at a ratio of approximately 1 molecule of Vif for every 75 to 220 molecules of p24, or 7 to 20 molecules per virion. Furthermore, we demonstrate that the relative concentration of Vif is much lower in particles than in infected cells, whereas the opposite is observed for the main virus components. The viral envelope, Nef, Vpr, Vpu, protease, reverse transcriptase, integrase, nucleocapsid, and p6gag proteins as well as the viral genomic RNA are dispensable for Vif packaging. Furthermore, mutating several highly conserved residues (H-108, C-114, C-133, L-145, and Q-146) or deleting the C-terminal 18 amino acids of Vif, either of which severely impairs Vif function, does not abolish its incorporation into virions. Finally, Vif can be packaged into murine leukemia virus particles. On the basis of these data, we conclude that the specificity of Vif incorporation into virions remains an open question.


Subject(s)
Gene Products, vif/metabolism , HIV-1/physiology , Viral Proteins/metabolism , Amino Acid Sequence , Blotting, Western , Cell Line , Conserved Sequence , Gene Products, vif/biosynthesis , Gene Products, vif/isolation & purification , Humans , Kidney , Kinetics , Mutagenesis, Site-Directed , RNA, Viral/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Sequence Deletion , T-Lymphocytes , Transfection , Viral Proteins/isolation & purification , Virion/physiology , vif Gene Products, Human Immunodeficiency Virus
5.
Methods Mol Biol ; 39: 295-315, 1995.
Article in English | MEDLINE | ID: mdl-7542523

Subject(s)
AIDS Vaccines , Cloning, Molecular/methods , Genetic Vectors , HIV Antigens/isolation & purification , HIV-1/genetics , HIV-2/genetics , Nucleopolyhedroviruses/genetics , Recombinant Fusion Proteins/isolation & purification , Animals , Base Sequence , Cell Line , Gene Products, env/biosynthesis , Gene Products, env/genetics , Gene Products, env/isolation & purification , Gene Products, gag/biosynthesis , Gene Products, gag/genetics , Gene Products, gag/isolation & purification , Gene Products, vif/biosynthesis , Gene Products, vif/genetics , Gene Products, vif/isolation & purification , HIV Antigens/biosynthesis , HIV Antigens/genetics , HIV Antigens/immunology , HIV Envelope Protein gp120/biosynthesis , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/isolation & purification , HIV Envelope Protein gp41/biosynthesis , HIV Envelope Protein gp41/genetics , HIV Envelope Protein gp41/isolation & purification , HIV Reverse Transcriptase , HIV-1/immunology , HIV-2/immunology , Molecular Sequence Data , Protein Precursors/biosynthesis , Protein Precursors/genetics , Protein Precursors/isolation & purification , RNA-Directed DNA Polymerase/biosynthesis , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Spodoptera , env Gene Products, Human Immunodeficiency Virus , gag Gene Products, Human Immunodeficiency Virus , vif Gene Products, Human Immunodeficiency Virus
6.
J Virol ; 68(2): 704-12, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8289374

ABSTRACT

The Vif (viral infectivity factor) protein of human immunodeficiency virus type 1 (HIV-1) has been shown to dramatically enhance the infectivity of HIV-1 virus particles during virus production. The subcellular localization of Vif was examined to elucidate cellular pathways which may be important for Vif function. Indirect immunofluorescence staining of Vif demonstrated a diffuse cytoplasmic distribution and showed that most Vif was not associated with the Golgi complex, a proposed site of localization (B. Guy, M. Geist, K. Dott, D. Spehner, M.-P. Kieny, and J.-P. Lecocq, J. Virol. 65:1325-1331, 1991). Subcellular fractionation of transfected COS cells and HIV-1-infected Jurkat and CEM cells demonstrated that Vif is a cytoplasmic protein which exists in both a soluble cytosolic form and membrane-associated form. The membrane-associated form of Vif is a peripheral membrane protein which is tightly associated with the cytoplasmic side of cellular membranes. The C terminus of Vif was required for the stable association of Vif with membranes. The C terminus was also essential for Vif function, suggesting that the association of Vif with membranes is likely to be important for its biological activity. The highly conserved regions at residues 103 to 115 and 142 to 150 were important for Vif function but did not affect membrane association, indicating that these regions are likely to be important for other, as-yet-unknown functions.


Subject(s)
Cell Compartmentation , Gene Products, vif/isolation & purification , HIV-1/chemistry , Membrane Proteins/isolation & purification , Animals , Biological Transport , Cells, Cultured , Cytosol/chemistry , DNA Mutational Analysis , Fluorescent Antibody Technique , Genetic Complementation Test , HIV-1/growth & development , Sequence Deletion , Subcellular Fractions/chemistry , Virus Replication , vif Gene Products, Human Immunodeficiency Virus
7.
AIDS Res Hum Retroviruses ; 9(10): 1025-30, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8280477

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) vif gene (viral infectivity gene) plays an important role in viral replication in vitro. We demonstrated that the Vif protein is membrane associated in HIV-1-infected cells and have investigated the role in viral replication of the equivalent gene in HIV-2. We constructed an HIV-2 vif minus mutant and studied its virulence and cellular tropism in vitro. Parallel experiments were also performed with an HIV-1 vif mutant to ascertain whether the two distantly related HIV-2 and HIV-1 genes might exert the same effect on viral replication. The results indicated that both HIV-1 and HIV-2 vif minus cell-free infection was not impaired when the SupT-1 cell line was used. However, differential degrees of impairment in viral replication were observed when other cell lines were used (Molt-3, U-937). Nevertheless, when viral production could not be detected, rescue experiments by coculture with the permissive cell line SupT-1 were generally positive, indicating that the viruses were still present in the inoculated cells. In contrast, when primary human cells (peripheral blood mononuclear cells and purified macrophages) were infected with HIV-1 and HIV-2 vif minus viruses no productive infection was observed and generally no virus was rescued by cocultivation. Thus, like in HIV-1, the vif gene of HIV-2 is crucial for viral infectivity in primary cells and might represent an attractive target for therapy.


Subject(s)
Cell Compartmentation , Cytoplasm/chemistry , Gene Products, vif/isolation & purification , Genes, vif , HIV/growth & development , Cell Fractionation , Cell Line , Gene Deletion , Gene Products, vif/genetics , HIV/genetics , HIV/pathogenicity , HIV-1/genetics , HIV-1/growth & development , HIV-1/pathogenicity , HIV-2/genetics , HIV-2/growth & development , HIV-2/pathogenicity , Humans , T-Lymphocytes/microbiology , Virulence , Virus Replication , vif Gene Products, Human Immunodeficiency Virus
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