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

描述(申请人提供)：我们和其他人最近的研究基本上阐明了HIV-1编码的病毒传染性因子(Vif)在中和发生在淋巴细胞和巨噬细胞以及一些白血病T细胞系中的强大抗病毒系统中的作用。这个抗病毒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，在那里它们对新合成的病毒逆转录本进行致命的高突变。VIF与A3G和ASF结合，并诱导它们的多泛素化和降解，从而将它们从感染细胞中消除，并阻止它们进入HIV-1后代。虽然研究人员使用了一种VIF作为标准，但HIV-1 VIF是高度不同的，我们发现自然HIV-1分离株编码的VIF与所有APOBEC3并列蛋白混杂结合，但对它们的浓度有很大不同的影响。此外，APOBECs在HIV-1易感细胞中以不同的数量和比例共表达，它们广泛地异源聚合形成一个协同和可诱导的抗病毒网络。我们的结果提示，Vif的多样性部分是对不同细胞和隔室中APOBEC3网络多样性的适应性反应，它可能在HIV-1的发病机制中发挥关键作用，在抗Vif药物的开发中也应该考虑。基于这些结果和大量的初步证据，我们提出了三个实质性和协同性的目标：(1)开发一系列强大的筛选平台，用于鉴定抑制哺乳动物细胞内不同vif的小分子。(2)我们发现HIV-1 Vif是在酵母中作为一种与A3G相关的可溶性蛋白而产生的。在毕赤酵母中产生大量的Vif及其A3G结合亚区和A3G，以此为资源分析抑制因子的作用机制，并支持AIM 3的高通量筛选体系。(3)优化ELISA法，筛选和分析抑制Vif-A3G结合的化合物，并测定不同VIF与A3G和其他胞苷脱氨酶的亲和力。该计划建立在最新的见解基础上，以开发和优化艾滋病的新的抑制剂筛选方法和研究资源，作为有效的抗VIF疗法的高通量筛选的基本前奏。

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.