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

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的强效抗逆转录病毒系统。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中。 1个核心，在那里它们致命地使新合成的病毒逆转录物超突变。Vif与A3 G结合， A3 F并诱导其多聚泛素化和降解，从而将其从感染的细胞中消除， 从而阻止它们掺入HIV-1后代中。额外的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，并且据信它们的浓度也可能 在T细胞和巨噬细胞中相对较低。然而，新的证据表明，APOBEC 3表达 细胞外信号和细胞特异性因子可显著改变其水平和活性。 因此，这些其他APOBEC 3旁系同源物可能潜在地影响HIV-1在特定细胞中的复制， 隔室或在一些患者组织中的炎症期间。最近，我们发现HIV-1病毒感染者 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变体降解 在标准测定系统中具有高度独特特异性的多种APOBECS。Vif中的几个突变 消除其与A3 G的结合并不消除其与ASF的结合，这表明这些关键的胞苷 脱氨酶与Vif的关联不同。这些以及最近的其他见解提出了重要的问题 关于Vif与ASF和A3 G结合的机制，Vif多样性在患者中的作用， 与A3 G和ASF相关的控制其抗HIV-1的细胞因子的身份和功能 活动此外，我们最近成功地在毕赤酵母中生产了大量Vif和A3 G pastoris酵母，这使得结构调查。基于这些考虑，我们提出三个 实质性和协同性目标：（1）优化可溶性Vif，A3 G， 的ASF。分析Vif同源寡聚化，确定Vif是否与A3 G和/或ASF直接缔合， 并将这些纯化的蛋白质用于协同的物理和结构研究。现场指导 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谱 光谱法来鉴定与A3 G相关的细胞蛋白质和RNA，并分析这些蛋白质和RNA的作用。 A3 G和ASF的抗HIV-1活性的蛋白质和RNA。该方案实质性地解决了重要的 有关Vif和APOBEC 3的问题，并提供了独特的方法和资源， 分析它们在HIV-1复制和发病机制中的作用。

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.