Roles of Vif Variants and APOBEC3s in HIV-1/AIDS

Vif 变体和 APOBEC3 在 HIV-1/AIDS 中的作用

• 批准号: 7232104
• 负责人: David Kabat
• 金额: $ 32.45万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7232104
• 关键词: Address; Affinity Chromatography; Anti-Retroviral Agents; Antiviral Agents; Binding; Binding Sites; Biological Assay; Cell Line; Cell physiology; Cells; Cultured Cells; Cytidine Deaminase; HIV; HIV-1; Human; Immunofluorescence Microscopy; Inflammation; Investigation; Lymphocyte; Mass Spectrum Analysis; Modeling; Mutation; Pathogenesis; Patients; Pichia; Polyubiquitination; Production; Proteins; RNA-Binding Proteins; Recombinants; Resources; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Specificity; Standards of Weights and Measures; Subgroup; System; T-Lymphocyte; Tissues; Transcript; Variant; Viral; Virus; Yeasts; acrosome stabilizing factor; base; extracellular; in vivo; insight; macrophage; paralogous gene; programs

DESCRIPTION (provided by applicant): Recent studies have begun to elucidate the role of the HIV-1 encoded viral infectivity factor (Vif) in neutralizing a potent antiretroviral system that occurs in lymphocytes and macrophages. This system principally involves the cytidine deaminases APOBEC3G (A3G) and/or ASF, which are incorporated into HIV- 1 cores where they lethally hypermutate newly synthesized viral reverse transcripts. Vif binds to A3G and A3F and induces their polyubiquitination and degradation, thereby eliminating them from infected cells and precluding their incorporation into HIV-1 progeny. The additional APOBEC3 paralogs ASA, A3B, and A3C have weaker anti-HIV-1 activities in standard assays, and it is believed that their concentrations might also be relatively low in T cells and macrophages. However, new evidence suggests that APOBEC3 expression levels and activities can be altered dramatically by extracellular signals and by cell-specific factors. Therefore, these other APOBEC3 paralogs might potentially influence HIV-1 replication in specific cells or compartments or during inflammation in some patient tissues. Recently, we found that HIV-1 Vifs use nonidentical sites to promiscuously bind to all APOBEC3 paralogs and that natural Vif variants degrade diverse APOBECSs with highly distinctive specificities in a standard assay system. Several mutations in Vif that eliminate its binding to A3G do not eliminate its binding to ASF, suggesting that these key cytidine deaminases associate differently with Vif. These and additional recent insights raise important questions concerning the mechanisms for Vif binding to ASF and A3G, the roles of Vif diversity in patients, and the identities and functions of cellular factors that associate with A3G and ASF to control their anti-HIV-1 activities. In addition, we have recently succeeded in producing large amounts of Vif and A3G in Pichia pastoris yeast, which enables structural investigations. Based on these considerations, we propose three substantive and synergistic aims: (1) Optimize large-scale production and purification of soluble Vif, A3G, and ASF. Analyze Vif homooligomerization, determine whether Vif associates directly with A3G and/or ASF, and use these purified proteins in collaborative physical and structural investigations. Employ site-directed mutagenesis to further analyze these protein interactions. (2) Analyze the role of adaptive Vif variance in HIV-1 replication in patients and in cell cultures. (3) Use tandem affinity purification and electrospray mass spectrometry to identify cellular proteins and RNAs that associate with A3G, and analyze the effects of these proteins and RNAs on anti-HIV-1 activities of A3G and ASF. This program substantively addresses important issues concerning Vif and APOBEC3 diversities and provides unique approaches and resources for analyzing their roles in HIV-1 replication and pathogenesis.

描述（由申请人提供）：最近的研究已经开始阐明HIV-1编码的病毒感染因子（Vif）在中和淋巴细胞和巨噬细胞中存在的强效抗逆转录病毒系统中的作用。该系统主要涉及胞苷脱氨酶APOBEC 3G（A3 G）和/或ASF，它们被掺入HIV- 1核心，在那里它们致命地超突变新合成的病毒逆转录物。Vif与A3 G和A3 F结合并诱导它们的多聚泛素化和降解，从而将它们从感染的细胞中消除并阻止它们掺入HIV-1后代。另外的APOBEC 3旁系同源物阿萨、A3 B和A3 C在标准测定中具有较弱的抗HIV-1活性，并且据信它们在T细胞和巨噬细胞中的浓度也可能相对较低。然而，新的证据表明，APOBEC 3的表达水平和活性可以通过细胞外信号和细胞特异性因子显着改变。因此，这些其他APOBEC 3旁系同源物可能会影响HIV-1在特定细胞或隔室中或在某些患者组织中的炎症期间的复制。最近，我们发现HIV-1 Vif使用不相同的位点混杂地结合所有APOBEC 3旁系同源物，并且天然Vif变体在标准测定系统中以高度独特的特异性降解不同的APOBECSs。Vif中的几个突变消除了其与A3 G的结合，但并没有消除其与ASF的结合，这表明这些关键的胞苷脱氨酶与Vif的结合不同。这些和其他最近的见解提出了重要的问题，有关的机制为Vif结合ASF和A3 G，Vif的多样性在患者中的作用，以及与A3 G和ASF控制其抗HIV-1活性的细胞因子的身份和功能。此外，我们最近成功地在巴斯德毕赤酵母中生产了大量的Vif和A3 G，这使得结构研究成为可能。基于这些考虑，我们提出了三个实质性的协同目标：（1）优化可溶性Vif，A3 G和ASF的大规模生产和纯化。分析Vif同源寡聚化，确定Vif是否与A3 G和/或ASF直接相关，并将这些纯化的蛋白质用于协同的物理和结构研究。采用定点突变进一步分析这些蛋白质的相互作用。(2)分析适应性Vif变异在患者和细胞培养中HIV-1复制中的作用。(3)利用串联亲和纯化和电喷雾质谱技术鉴定与A3 G相关的细胞蛋白和RNA，并分析这些蛋白和RNA对A3 G和ASF抗HIV-1活性的影响。该计划实质性地解决了有关Vif和APOBEC 3缺陷的重要问题，并为分析它们在HIV-1复制和发病机制中的作用提供了独特的方法和资源。