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

最近的研究已经开始阐明HIV-1编码的病毒感染性因子(Vif)在 中和发生在淋巴细胞和巨噬细胞中的强大的抗逆转录病毒系统。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，它们致命地高突变新合成的病毒逆转录本。VIF绑定到A3G和 A3F并诱导它们的多泛素化和降解，从而将它们从感染的细胞和 排除它们与HIV-1后代的结合。附加的APOBEC3并列表ASA、A3B和A3C 在标准检测中具有较弱的抗HIV-1活性，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APOBEC3的表达 细胞外信号和细胞特异性因子可以极大地改变细胞水平和活性。 因此，这些APOBEC3同源基因可能会潜在地影响HIV-1在特定细胞或 在某些患者组织内或在炎症期间。最近，我们发现HIV-1 vif使用 不完全相同的位点混杂地与所有APOBEC3并列基因结合，并且天然的Vif变体降级 在一个标准的检测系统中，具有高度独特的特异性的各种APOBEC。Vif基因的几个突变 消除其与A3G结合并不会消除其与ASF的结合，这表明这些关键的胞苷 脱氨酶与VIF有不同的关联。这些和最近的其他见解提出了重要的问题 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 调控A3G和ASF抗HIV-1的细胞因子的特性和功能 活动。此外，我们最近在毕加索成功地生产了大量的VIF和A3G 巴斯德酵母，这使得结构研究成为可能。基于这些考虑，我们提出了三点建议。 实质性和协同性目标：(1)优化大规模生产和纯化可溶性VIF、A3G、 和ASF。分析VIF同源齐聚，确定VIF是否直接与A3G和/或ASF结合， 并将这些纯化的蛋白质用于合作的物理和结构研究。采用现场定向 进一步分析这些蛋白质的相互作用。(2)分析了自适应VIF方差在VIF模型中的作用 HIV-1在患者体内和细胞培养中的复制。(3)采用串联亲和纯化和电喷雾质量法 以确定与A3G相关的细胞蛋白质和RNA，并分析这些影响 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的 有关VIF和APOBEC3多样性的问题，并提供了独特的方法和资源 分析它们在HIV-1复制和致病机制中的作用。

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.