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

Vif 变体和 APOBEC3 在 HIV-1/AIDS 中的作用

基本信息

批准号：
7414558
负责人：
David Kabat
金额：
$ 33.01万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-07-01 至 2010-04-30
项目状态：
已结题

项目摘要

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 intoHIV- 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.

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有效抗逆转录病毒系统。这个系统主要涉及胞苷脱氨酶 APOBEC3G (A3G) 和/或 ASF，它们被整合到 HIV- 1 个核心，它们对新合成的病毒逆转录物进行致命性超突变。 Vif 与 A3G 结合并且 A3F 并诱导它们的多泛素化和降解，从而将它们从受感染的细胞中消除，阻止它们融入 HIV-1 后代。其他 APOBEC3 旁系同源物 ASA、A3B 和 A3C 在标准测定中具有较弱的抗 HIV-1 活性，据信它们的浓度也可能 T 细胞和巨噬细胞含量相对较低。然而，新的证据表明 APOBEC3 表达细胞外信号和细胞特异性因素可以显着改变水平和活性。因此，这些其他 APOBEC3 旁系同源物可能会影响特定细胞中的 HIV-1 复制或隔室或某些患者组织炎症期间。最近，我们发现 HIV-1 Vifs 使用与所有 APOBEC3 旁系同源物混杂结合的不同位点，并且天然 Vif 变体会降解在标准检测系统中具有高度独特特异性的多种 APOBECS。 Vif 的几个突变消除其与 A3G 的结合并不能消除其与 ASF 的结合，表明这些关键胞苷脱氨酶与 Vif 的关联方式不同。这些和其他最新见解提出了重要问题关于 Vif 与 ASF 和 A3G 结合的机制、Vif 多样性在患者中的作用以及与 A3G 和 ASF 相关的细胞因子的特性和功能，以控制其抗 HIV-1 活动。另外，我们最近在毕赤酵母中成功大量生产了Vif和A3G 巴斯德酵母，可以进行结构研究。基于这些考虑，我们提出三点建议实质性和协同目标：（1）优化可溶性Vif、A3G、和非洲猪瘟。分析 Vif 同聚，确定 Vif 是否直接与 A3G 和/或 ASF 结合，并在协作物理和结构研究中使用这些纯化的蛋白质。采用现场定向诱变以进一步分析这些蛋白质相互作用。 (2)分析自适应Vif方差在 HIV-1 在患者和细胞培养物中的复制。 (3) 使用串联亲和纯化和电喷雾质量光谱法来鉴定与 A3G 相关的细胞蛋白和 RNA，并分析这些蛋白和 RNA 的影响蛋白质和 RNA 对 A3G 和 ASF 抗 HIV-1 活性的影响。该计划实质上解决了重要问题有关 Vif 和 APOBEC3 多样性的问题，并提供独特的方法和资源分析它们在 HIV-1 复制和发病机制中的作用。