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

Vpr（病毒蛋白R）是一种由HIV-1编码的辅助蛋白， 1，几十年来一直是个谜。Vpr导致细胞周期停滞在G2/M期，引发DNA损伤 反应，并增强病毒基因表达。它通过将宿主 用于降解、劫持基于cullin 4的E3泛素连接酶复合物（CRL 4）以诱导 他们的消耗。我们最近发现了一种宿主蛋白CCDC 137，也称为cPERP-B， 以CRL 4复合物依赖性方式被Vpr耗尽的关键靶蛋白。特别是，CCDC 137 通过RNA干扰的消减再现了Vpr对宿主和病毒的上述作用。 本课题旨在研究CCDC 137抑制HIV-1基因的分子机制 表达以及它如何控制细胞周期进程和DNA损伤反应。在 目的1，我们将确定CCDC 137缺失是否是Vpr蛋白的保守特征， 不同的HIV和SIV毒株，绘制Vpr诱导耗竭所需的CCDC 137决定簇， 并评估来自不同病毒的Vpr对病毒基因表达的影响。此外，我们将 定义Vpr消耗CCDC 137所需的宿主蛋白。目标2以 CCDC 137介导的HIV-1基因表达抑制机制。我们会描述顺- CCDC 137介导的抑制所需的作用序列，并评估 整合和整合位点选择对Vpr/CCDC 137调节的HIV-1基因表达的影响。 我们还将结合联合收割机筛选方法（蛋白质组学、酵母双杂交和CRISPR功能 筛选）以鉴定CCDC 137相互作用辅因子，以阐明 CCDC 137抑制HIV-1基因表达。在目标3中，我们将研究CCDC 137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 CCDC 137是否保护染色体DNA并描述与CCDC 137协同作用的宿主因子， CCDC 137调节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.