Structure and Function of APOBEC Proteins

我们将描述HIV-1 Vif、HIV-2 Vif、A3G和A3F的结构决定因素，它们相互作用并导致A3G和A3F的降解。与A3G和A3F相互作用的HIV-1 Vif的决定因素在HIV-2 Vif中并不保守，表明HIV-2 Vif通过不同的相互作用诱导A3蛋白的降解。我们将对HIV-1 Vif、HIV-2 Vif、A3G和A3F进行广泛的突变分析，以确定每个蛋白质相互作用的结构决定因素。我们还将使用双分子荧光互补来表征细胞中A3G-A3G相互作用的决定因素。我们将开发A3G-Vif和A3F-Vif相互作用的小分子抑制剂。我们开发了高通量原位检测方法，用于高通量筛选干扰HIV-1 Vif介导的A3G和A3F降解的小分子抑制剂。与美国国立卫生研究院化学基因组学中心合作，我们将筛选一个包含300,000多种化合物的文库，以确定抗病毒药物开发的这一新靶点的抑制剂。我们还将确定HIV-2、Vif和A3相互作用的抑制剂，用于治疗HIV-2感染。我们将确定A3G和A3F介导的超突变对HIV-1基因组的影响。为了深入了解高突变对病毒进化的影响，我们将进行超深度测序分析，以相对于A3介导的G-to-A高突变靶点识别和表征整个HIV-1基因组，以确定突变热点和冷点。我们将确定A3F上泛素化的位置。我们将量化A3蛋白在原代CD4+T细胞和巨噬细胞中的抗病毒活性。我们将使用不同的激活模式和细胞因子刺激来测定A3G和A3F在原代激活的CD4+T细胞和巨噬细胞中的相对抑制潜力，以了解它们在不同生理条件下的抗病毒活性。我们将分析A3蛋白在P小体中的作用，以及Mov10抑制逆转录病毒复制的机制。我们观察到，Mov10是一种干扰素诱导的P体蛋白，在病毒产生和逆转录水平上抑制HIV-1的复制。为了阐明抑制机制，我们将分析Mov10病毒粒子的掺入及其对Gag表达和反转录的影响。[对应于2007年4月艾滋病毒耐药计划现场访问报告中的Pathak项目1]

We will characterize structural determinants of HIV-1 Vif, HIV-2 Vif, A3G, and A3F that interact and induce degradation of A3G and A3F. The determinants of HIV-1 Vif that interact with A3G and A3F are not conserved in HIV-2 Vif, suggesting that HIV-2 Vif induces A3 protein degradation using distinct interactions. We will carry out extensive mutational analysis of HIV-1 Vif, HIV-2 Vif, A3G, and A3F to identify structural determinants of each protein that interact with each other. We will also characterize determinants of A3G-A3G interactions in cells using bimolecular fluorescence complementation. We will develop small-molecule inhibitors of A3G-Vif and A3F-Vif interactions. We have developed in situ assays for high-throughput screening of small-molecule inhibitors that interfere with HIV-1 Vif-mediated degradation of A3G and A3F. In collaboration with the NIH Chemical Genomics Center, we will screen a library of greater than 300,000 compounds to identify inhibitors of this novel target for antiviral drug development. We will also identify inhibitors of HIV-2 Vif and A3 interaction for the treatment of HIV-2 infection. We will determine the impact of A3G- and A3F-mediated hypermutation on the HIV-1 genome. To gain insight into the impact of hypermutation on viral evolution, we will carry out ultradeep sequencing analysis to identify and characterize the entire HIV-1 genome with respect to A3-mediated G-to-A hypermutation target sites to identify mutational hotspots and coldspots. We will identify the site of ubiquitination on A3F. We will quantify the antiviral activity of A3 proteins in primary CD4+ T cells and macrophages. We will determine the relative inhibitory potential of A3G and A3F in primary activated CD4+ T cells and macrophages using different modes of activation and cytokine stimulation to gain insights into their antiviral activity under various physiological conditions. We will analyze the role of A3 proteins in P bodies, and the mechanism by which Mov10 inhibits retroviral replication. We have observed that Mov10, an interferon-inducible P body protein, inhibits HIV-1 replication at the level of virus production and reverse transcription. To elucidate the mechanism of inhibition, we will analyze Mov10 virion incorporation and its effects on Gag expression and reverse transcription. [Corresponds to Pathak Project 1 in the April 2007 site visit report of the HIV Drug Resistance Program]