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ABSTRACT: HIV-1 Vif protein protects viral replication in non-permissive cells by inducing degradation of APOBEC3G via ubiquitination and proteasomal pathway, although new studies indicate a putative role in Vif's direct inhibition of APOBEC3G. APOBEC3G is member of a homologous family of proteins with cytidine deaminase activity expressed with characteristic tissue specificity, that in humans consist of APOBEC1, APOBEC2, APOBEC3A-H, APOBEC4 and the activation-induced deaminase (AID), a B lymphoid protein necessary for somatic hypermutation, gene conversion and class switch recombination. In this work we show that Vif can counteract AID's activity in E. coli in absence of specific eukaryotic co-factors necessary for AID induced somatic hypermutation, gene conversion and to stimulate class switch recombination in B-cells. We show that AID inhibition is mediated by a direct protein-protein interaction via unique amino acid D118 an homologous mutant responsible for the species-specific restriction of HIV-1 Vif protein existent for APOBEC3G. These results raise the hypothesis that Vif related proteins can act as a broad inhibitor of deaminase activity. Moreover as AID and Vif evolved in different cellular environments, these results may indicate that Vif related proteins might mimic cellular factors that interact with a structural conserved domain of cytidine deaminases during evolution.
Molecular Immunology 02/2007; 44(4):583-90. · 2.90 Impact Factor
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ABSTRACT: The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G), also known as CEM-15, is a host-cell factor involved in innate resistance to retroviral infection. HIV-1 viral infectivity factor (Vif) protein was shown to protect the virus from APOBEC3G-mediated viral cDNA hypermutation. The mechanism proposed for protection of the virus by HIV-1 Vif is mediated by APOBEC3G degradation through ubiquitination and the proteasomal pathway. Here we show that in Escherichia coli the APOBEC3G-induced cytidine deamination is inhibited by expression of Vif without depletion of deaminase. Moreover, inhibition of deaminase-mediated bacterial hypermutation is dependent on a single amino acid substitution D128K that renders APOBEC3G resistant to Vif inhibition. This single amino acid was elegantly proven by other authors to determine species-specific sensitivity. Our results show that in bacteria this single amino acid substitution controls Vif-dependent blocking of APOBEC3G that is dependent on a strong protein interaction. The C-terminal region of Vif is responsible for this strong protein-protein interaction. In conclusion, our experiments suggest a complement to the model of Vif-induced degradation of APOBEC3G by bringing to relevance that deaminase inhibition can also result from a direct interaction with Vif protein.
Journal of Biological Chemistry 04/2005; 280(10):8765-75. · 4.77 Impact Factor
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ABSTRACT: 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.
Journal of Virology 02/2005; 79(2):823-33. · 5.40 Impact Factor
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ABSTRACT: HIV-1 Vif (viral infectivity factor) protein overcomes the antiviral activity of the DNA deaminase APOBEC3G by targeting it for proteasomal degradation. We report here that Vif targets APOBEC3G for degradation by forming an SCF-like E3 ubiquitin ligase containing Cullin 5 and Elongins B and C (Cul5-EloB-EloC) through a novel SOCS (suppressor of cytokine signaling)-box that binds EloC. Vif binding to EloC is negatively regulated by serine phosphorylation in the BC-box motif of the SOCS-box. Vif ubiquitination is promoted by Cul5 in vitro and in vivo, and requires an intact SOCS-box. Thus, autoubiquitination of Vif occurs within the assembled Vif-Cul5 complex, analogous to F-box proteins that are autoubiquitinated within their SCF (Skp1-Cullin-F-box) complex. These findings suggest mechanisms that regulate the assembly and activity of Cul5 E3 complexes through phosphorylation or autoubiquitination of the SOCS-box protein, and identify interactions between Vif and host cell proteins that may be therapeutic targets.
Genes & Development 01/2005; 18(23):2861-6. · 11.66 Impact Factor
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Journal of Biological Chemistry. 12/2004;
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ABSTRACT: We recently developed a specific single-chain antibody from immunized rabbits to HIV-1 Vif protein that was expressed intracellularly and inhibited reverse transcription and viral replication. The Vif of HIV-1 overcomes the innate antiviral activity of a cytidine deaminase Apobec3G (CEM15) that induces G to A hypermutation in the viral genome, resulting in enhancement of viral replication infectivity. Here, we have developed a minimal scaffold VH fragment with intrabody properties derived from anti-Vif single-chain antibody that was engineered to mimic camelid antibody domains. Non-specific binding of VH by its interface for the light chain variable domain (VL) was prevented through amino acid mutations in framework 2 and 4 (Val37F, G44E, L45R, W47G and W103R). Our results demonstrate that all constructed anti-Vif VH single-domains preserve the antigen-binding activity and specificity in the absence of the parent VL domain. However, only the most highly camelized domains had high levels of intracellular expression. The expression in eukaryotic cells showed that VH single-domains could correctly fold as soluble proteins in the reducing environment. The results demonstrated an excellent correlation between improvements in protein solubility with gradually increasing camelization. Camelized single-domains efficiently bound Vif protein and neutralized its infectivity enhancing function, by reducing late reverse transcripts and proviral integration. The activity of the anti-Vif single-domains was shown to be cell-specific, with inhibitory effects only in cells non-permissive that require Vif for HIV-1 replication. Moreover, cell specificity of anti-Vif intrabodies was correlated with an increase of Apobec3G, which potentiates viral inhibition. The present study strongly suggests that camelization of rabbit VH domains is a potentially useful approach for engineering intrabodies for gene therapy.
Journal of Molecular Biology 08/2004; 340(3):525-42. · 4.00 Impact Factor
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ABSTRACT: 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.
Retrovirology 02/2004; 1:28. · 6.47 Impact Factor
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ABSTRACT: Human immunodeficiency virus type 1 (HIV-1)-encoded Vif protein is important for viral replication and infectivity. Vif is a cytoplasmic protein that acts during virus assembly by an unknown mechanism, enhancing viral infectivity. The action of Vif in producer cells is essential for the completion of proviral DNA synthesis following virus entry. Therefore, Vif is considered to be an important alternative therapeutic target for inhibition of viral infectivity at the level of viral assembly and reverse transcription. To gain insight into this process, we developed a Vif-specific single-chain antibody and expressed it intracellularly in the cytoplasm. This intrabody efficiently bound Vif protein and neutralized its infectivity-enhancing function. Intrabody-expressing cells were shown to be highly refractory to challenge with different strains of HIV-1 and HIV-1-infected cells. Inhibition of Vif by intrabody expression in the donor cell produced viral particles that do not complete reverse transcription in the recipient cell. The anti-Vif scFv was shown to be specific for Vif protein because its function was observed only in nonpermissive cells (H9, CEM, and U38). Moreover, transduction of peripheral blood mononuclear cells with an HIV-derived retroviral vector expressing Vif intrabody was shown to confer resistance to laboratory-adapted and primary HIV strains. This study provides biochemical evidence for the role of Vif in the HIV-1 lifecycle and validates Vif as a target for the control of HIV-1 infection.
Journal of Biological Chemistry 09/2002; 277(35):32036-45. · 4.77 Impact Factor
