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

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发生的有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APOBEC3G （A3G）和/或ASF，它们被整合到HIV- 1核心中，在那里它们致命地高突变新合成的病毒逆转录物。Vif与A3G和A3F结合，诱导它们的多泛素化和降解，从而将它们从感染细胞中清除，并阻止它们并入HIV-1后代。另外的APOBEC3类似物ASA、A3B和A3C在标准检测中具有较弱的抗hiv -1活性，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，APOBEC3的表达水平和活性可以被细胞外信号和细胞特异性因子显著改变。因此，这些其他APOBEC3类似物可能潜在地影响HIV-1在某些患者组织的特定细胞或区室或炎症期间的复制。最近，我们发现HIV-1 Vif使用不相同的位点来混杂结合所有APOBEC3类似物，并且在标准测定系统中，天然Vif变体以高度不同的特异性降解多种apobecs。Vif中的一些突变消除了它与A3G的结合，但没有消除它与ASF的结合，这表明这些关键的胞苷脱氨酶与Vif的关联不同。这些和最近的其他见解提出了关于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相关的细胞因子的身份和功能以控制其抗hiv -1活性的重要问题。此外，我们最近成功地在毕氏酵母中生产了大量的Vif和A3G，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、A3G和ASF的大规模生产和纯化。分析Vif的同质寡聚化，确定Vif是否与A3G和/或ASF直接相关，并将这些纯化蛋白用于协同物理和结构研究。利用定点诱变进一步分析这些蛋白质相互作用。(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)采用串联亲和纯化和电喷雾质谱技术鉴定与A3G相关的细胞蛋白和rna，分析这些蛋白和rna对A3G和ASF抗hiv -1活性的影响。该项目实质性地解决了有关Vif和APOBEC3多样性的重要问题，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。

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.