Drug development for Vif-APOBEC3G in HIV-1/AIDS

描述（由申请人提供）：我们和其他人最近的研究已经基本阐明了HIV-1编码的病毒感染因子（Vif）在中和淋巴细胞和巨噬细胞以及一些白血病T细胞系中存在的强效抗病毒系统中的作用。这种抗病毒系统主要涉及胞苷脱氨酶APOBEC 3G（A3 G）和/或ASF，其被掺入HIV-1核心，在那里它们致命地超突变新合成的病毒逆转录物。Vif与A3 G和ASF结合并诱导它们的多聚泛素化和降解，从而将它们从感染的细胞中消除并阻止它们掺入HIV-1后代。尽管研究人员使用一种Vif作为标准，但HIV-1 Vif具有高度的差异性，我们发现天然HIV-1分离株编码的Vif与所有APOBEC 3旁系同源物混杂结合，但对其浓度具有广泛的独特影响。此外，APOBECS在HIV-1易感细胞中以不同的量和比例共表达，并且它们广泛地异源寡聚化以形成协作的和可诱导的抗病毒网络。我们的研究结果表明，Vif多样性部分是对不同细胞和隔室中APOBEC 3网络多样性的适应性反应，它可能在HIV-1发病机制中发挥关键作用，并且在抗Vif药物开发中也应考虑。基于这些结果和大量的初步证据，我们提出了三个实质性的和协同的目标：（1）开发一个强大的系列筛选平台，用于识别小分子抑制哺乳动物细胞内的各种VIFs。(2)我们发现HIV-1 Vif在酵母中作为与A3 G相关的可溶性蛋白质产生。在毕赤酵母中产生大量Vif及其A3 G结合亚结构域和A3 G，作为分析抑制剂机制的资源，并支持目标3的高通量筛选系统。(3)优化ELISA测定以筛选和分析抑制Vif-A3 G结合的化合物，并测量不同Vif对A3 G和其他胞苷脱氨酶的亲和力。该计划建立在最近的见解，开发和优化新的抑制剂筛选方法和艾滋病的研究资源，作为一个重要的前奏，高通量筛选有效的抗Vif治疗。

DESCRIPTION (provided by applicant): Recent studies by ourselves and others have substantially elucidated the role of the HIV-1 encoded viral infectivity factor (Vif) in neutralizing a potent antiviral system that occurs in lymphocytes and macrophages and some leukemic T cell lines. This antiviral 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 ASF and induces their polyubiquitination and degradation, thereby eliminating them from infected cells and precluding their incorporation into HIV-1 progeny. Although researchers have used one Vif as a standard, HIV-1 Vifs are highly divergent and we have found that natural HIV-1 isolates encode Vifs that bind promiscuously to all APOBEC3 paralogs but have widely distinctive effects on their concentrations. In addition, the APOBECSs are coexpressed in different amounts and proportions in HIV-1 susceptible cells, and they broadly heterooligomerize to form a collaborative and inducible antiviral network. Our results suggest that Vif diversity is partly an adaptive response to the diversity of the APOBEC3 network in different cells and compartments, that it may play a critical role in HIV-1 pathogenesis, and that it should also be considered in anti-Vif drug development. Based on these results and on substantial preliminary evidence, we propose three substantial and synergistic aims: (1) Develop a robust series of screening platforms for identification of small molecules that inhibit diverse Vifs within mammalian cells. (2) We found that HIV-1 Vif is made in yeast as a soluble protein that associates with A3G. Produce substantial amounts of Vif, and its A3G-binding subdomain and A3G in the yeast Pichia pastoris as a resource to analyze inhibitor mechanisms, and to support the high throughput screening system of aim 3. (3) Optimize ELISA assays to screen and analyze compounds that inhibit Vif- A3G binding and to measure affinities of diverse Vifs for A3G and other cytidine deaminases. This program builds on recent insights to develop and to optimize novel inhibitor screening approaches and investigational resources for AIDS, as an essential prelude to high throughput screening for effective anti-Vif therapeutics.