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.

最近的研究已开始阐明HIV-1编码的病毒感染因子（VIF）在中和发生在淋巴细胞和巨噬细胞中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，它们纳入了Inohiv- 1个核心，它们在致命的高充血新合成的病毒逆转录中。 VIF与A3G结合，并且 A3F并诱导它们的多泛素化和降解，从而从感染的细胞中消除了它们排除将其掺入HIV-1后代。附加的APOBEC3旁系同源物ASA，A3B和A3C 标准测定中的抗HIV-1活动较弱，据信他们的浓度也可能在T细胞和巨噬细胞中相对较低。但是，新的证据表明apobec3表达水平和活动可以通过细胞外信号和细胞特异性因素大大改变。因此，这些其他APOBEC3旁系同源物可能可能影响特定细胞中的HIV-1复制某些患者组织中的隔室或炎症期间。最近，我们发现HIV-1 VIF使用非相同的位点与所有apobec3旁系同源物结合，并且天然VIF变体降级在标准测定系统中具有高度独特特异性的不同apobecs。 VIF中的几个突变消除了其与A3G的结合并不能消除其与ASF的结合，这表明这些钥匙cytidine 脱氨酶与VIF不同。这些和最近的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 活动。此外，我们最近成功地在Pichia中生产了大量的VIF和A3G Pastoris酵母，可以进行结构研究。根据这些考虑，我们提出了三个实质和协同的目的：（1）优化可溶性VIF的大规模生产和纯化，A3G，和ASF。分析VIF家庭化，确定VIF是否与A3G和/或ASF直接关联并在协作的物理和结构研究中使用这些纯化的蛋白质。采用站点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方差在患者和细胞培养物中的HIV-1复制。（3）使用串联亲和力纯化和电喷雾质量识别与A3G相关的细胞蛋白和RNA的光谱法，并分析这些效果 A3G和ASF抗HIV-1活性的蛋白质和RNA。该计划实质上解决了重要有关VIF和APOBEC3多样性的问题，并为分析其在HIV-1复制和发病机理中的作用。