Structure and Function of APOBEC Proteins

我们将描述HIV-1 Vif，HIV-2 Vif，A3 G和A3 F相互作用并诱导A3 G和A3 F降解的结构决定因素。与A3 G和A3 F相互作用的HIV-1 Vif的决定簇在HIV-2 Vif中不保守，表明HIV-2 Vif使用不同的相互作用诱导A3蛋白降解。我们将对HIV-1 Vif、HIV-2 Vif、A3 G和A3 F进行广泛的突变分析，以确定每种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A3 G-A3 G相互作用的决定因素。 我们将开发A3 G-Vif和A3 F-Vif相互作用的小分子抑制剂。我们已经开发了高通量筛选干扰HIV-1 Vif介导的A3 G和A3 F降解的小分子抑制剂的原位测定。与NIH化学基因组学中心合作，我们将筛选一个超过30万种化合物的库，以确定这种新型靶点的抑制剂，用于抗病毒药物开发。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A3 G和A3 F介导的超突变对HIV-1基因组的影响。为了深入了解超突变对病毒进化的影响，我们将进行超深度测序分析，以确定和表征整个HIV-1基因组中A3介导的G到A超突变靶位点，以确定突变热点和冷点。 我们将确定A3 F上的泛素化位点。我们将量化A3蛋白在原代CD 4 + 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和细胞因子刺激模式来确定A3 G和A3 F在原代活化的CD 4 + T细胞和巨噬细胞中的相对抑制潜力，以深入了解它们在各种生理条件下的抗病毒活性。我们将分析A3蛋白在P体中的作用，以及Mov 10抑制逆转录病毒复制的机制。我们已经观察到，干扰素诱导的P体蛋白Mov 10在病毒产生和逆转录水平上抑制HIV-1复制。为了阐明抑制机制，我们将分析Mov 10病毒粒子掺入及其对Gag表达和逆转录的影响。 [对应于艾滋病毒耐药性方案2007年4月实地访问报告中的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]