Host protein targets of HIV-1 Vpr in gene expression, cell cycle and innate immunity

病毒蛋白R(Virus Protein R，VPR)是人类免疫缺陷病毒(HIV-1，HIV-1)编码的一种辅助蛋白。 1，几十年来一直是个谜。VPR导致细胞周期停滞于G2/M期，引发DNA损伤 应答，并增强病毒基因的表达。它通过以主机为目标来执行这些活动 蛋白质(S)降解、劫持基于cullin 4的E3泛素连接酶复合体(CRL4)诱导 它们的耗尽。我们最近发现了一种宿主蛋白CCDC137，也被称为cPERP-B，是一种 关键靶蛋白被VPR以CRL4复合体依赖的方式耗尽。具体来说，CCDC137 RNA干扰耗尽概括了前述VPR对宿主和病毒的影响。 在这个项目中，我们试图研究CCDC137如何抑制HIV-1基因的分子细节 表达以及它如何控制细胞周期进程和DNA损伤反应。在……里面 目的1，我们将确定CCDC137缺失是否是VPR蛋白的一个保守特征 不同的HIV和SIV毒株，绘制了VPR诱导的耗竭所需的CCDC137决定因素图， 并评估不同病毒的VPR对病毒基因表达的影响。此外，我们还将 确定VPR耗尽CCDC137所需的宿主蛋白。目标2的中心是 CCDC137抑制HIV-1基因表达的机制我们将划定独联体- CCDC137介导的抑制所需的作用序列和评价 整合及整合位点选择对VPR/CCDC137调控的HIV-1基因表达的影响 我们还将结合筛选方法(蛋白质组学、酵母双杂交和CRISPR功能 屏幕)，以确定CCDC137相互作用的辅助因子(S)，以阐明其机制 CCDC137抑制HIV-1基因表达。在目标3中，我们将调查CCDC137如何防止 DNA损伤反应和控制细胞周期进程。特别是，我们将确定 CCDC137是否保护染色体DNA并圈定宿主因素(S)配合 CCDC137调控DNA损伤反应。

The role and mechanism of action of Vpr (Viral Protein R), an accessory protein encoded by HIV- 1, has been enigmatic for decades. Vpr causes cell cycle arrest at G2/M, triggers a DNA damage response, and enhances viral gene expression. It exerts these activities by targeting host protein(s) for degradation, hijacking cullin4-based E3 ubiquitin ligase complex (CRL4) to induce their depletion. We recently identified a host protein CCDC137, also known as cPERP-B, as a key target protein depleted by Vpr in a CRL4 complex dependent manner. Specifically, CCDC137 depletion by RNA interference recapitulates the aforementioned effects of Vpr on host and virus. In this project we seek to study the molecular details of how CCDC137 represses HIV-1 gene expression as well as how it controls cell cycle progression and the DNA damage response. In Aim 1, we will determine whether CCDC137 depletion is a conserved feature of Vpr proteins from diverse HIV and SIV strains, map the CCDC137 determinants required for Vpr-induced depletion, and assess the effect of Vpr from diverse viruses on viral gene expression. In addition, we will define host proteins required for CCDC137 depletion by Vpr. Aim 2 is centered on the mechanisms of CCDC137-mediated repression of HIV-1 gene expression. We will delineate cis- acting sequences required for CCDC137-mediated repression and evaluate the effect of integration and integration site selection on the Vpr/CCDC137-regulated HIV-1 gene expression. We will also combine screening methods (proteomics, yeast 2-hybrid, and CRISPR functional screens) to identify CCDC137 interacting cofactor(s) to illuminate the mechanism of how CCDC137 inhibits HIV-1 gene expression. In Aim 3 we will investigate how CCDC137 prevents DNA damage response and controls cell cycle progression. In particular, we will determine whether CCDC137 protects chromosomal DNA and delineate host factor(s) cooperating with CCDC137 to modulate the DNA damage response.