Structure and Function of APOBEC3 Proteins

A3G、A3F和Mov10定位于被称为P小体的细胞质斑点，并抑制HIV-1的复制。我们探索了Mov10和A3蛋白P-小体定位的功能意义，发现它们的病毒粒子掺入或抗病毒活性不需要P-小体定位。我们进行了全基因组的siRNA筛选，以确定促进HIV-1 Vif(Vif1)介导的A3蛋白降解的宿主蛋白。筛选确定的一个因子是UBA52，一种泛素-核糖体蛋白L40融合蛋白；我们正在表征UBA52在Vif介导的A3蛋白降解中的作用。除了UBA52外，我们的全基因组siRNA筛选还发现了其他可能促进Vif1诱导的A3G降解的宿主因子，包括UBE4B、UBE3A、KAT5和WDR82。我们将研究这些和其他候选HIT的作用，以确定这些因素如何在Vif1存在的情况下促进A3蛋白的降解。Vif1和Vif2蛋白之间几乎没有同源性，我们最近的研究表明，Vif1和Vif2通过完全不同的决定因素与A3蛋白相互作用。我们目前的研究表明，Vif1和Vif2诱导A3蛋白降解的机制存在显著差异。我们将阐明Vif2介导的A3蛋白降解的机制。CBF-β是一种宿主因子，可与Vif1结合，增加其稳定性和抗病毒活性。我们观察到Vif2不与CBF-β相互作用。我们将进行免疫共沉淀和质谱学研究，以鉴定Vif2相互作用的蛋白，并确定是否有另一种宿主因子发挥与CBF-β相同的功能。HIV-1组N、O和P通过人畜传播的猿类免疫缺陷病毒感染人类。我们正在研究这些HIV-1 Vif蛋白与人类A3G和A3F蛋白之间的相互作用，并将它们与M组Vif的相互作用进行比较。虽然A3G、A3F和A3D在人的CD4T细胞中表达，但它们对限制Vif缺陷HIV-1的相对贡献尚不清楚。我们使用了在降解A3G或A3F/A3D方面存在缺陷的HIV-1 Vif突变体，发现A3G比A3F和A3D更有效地限制HIV-1。目前尚不清楚A3G和A3F能否并入相同的病毒粒子，并合并相同的HIV-1基因组。我们正在确定A3G和A3F并入相同的病毒粒子并合并HIV-1基因组的潜力。目前尚不清楚A3G和A3F诱导的突变是否会导致病毒基因变异和/或影响逆转录病毒重组的频率。我们正在进行实验，以确定A3G和A3F诱导的G-to-A突变是否有助于病毒的遗传多样性和进化。_[对应于2011年10月艾滋病毒耐药计划现场访问报告中的帕萨克项目2]

A3G, A3F, and Mov10 localize to cytoplasmic puncta known as P bodies and inhibit HIV-1 replication. We explored the functional significance of P-body localization for Mov10 and A3 proteins and found that P-body localization is not required for their virion incorporation or antiviral activity. We performed a genome-wide siRNA screen to identify host proteins that facilitate HIV-1 Vif (Vif1)-mediated degradation of A3 proteins. One factor that the screen identified is UBA52, a ubiquitin-ribosomal protein L40 fusion protein; we are characterizing the role of UBA52 in Vif-mediated degradation of A3 proteins. In addition to UBA52, our genome-wide siRNA screen identified other host factors that potentially facilitate Vif1-induced degradation of A3G, including UBE4B, UBE3A, KAT5, and WDR82. We will examine the role of these and other candidate hits to determine how these factors facilitate degradation of A3 proteins in the presence of Vif1. There is little homology between Vif1 and Vif2 proteins, and our recent studies indicate that Vif1 and Vif2 interact with A3 proteins by using completely different determinants. Our current studies suggest that there are significant differences in the mechanisms by which Vif1 and Vif2 induce degradation of A3 proteins. We will elucidate the mechanism of Vif2-mediated degradation of A3 proteins. CBF-beta is a host factor that binds to Vif1 and increases its stability and antiviral activity. We observed that Vif2 does not interact with CBF-beta. We will carry out coimmunoprecipitation and mass spectrometry studies to identify Vif2-interacting proteins and determine whether another host factor serves the same function as CBF-beta. HIV-1 groups N, O, and P have infected humans by zoonotic transmission of simian immunodeficiency viruses. We are characterizing the interactions between these HIV-1 Vif proteins and human A3G and A3F proteins and comparing their interactions with those of group M Vif. Although A3G, A3F, and A3D are expressed in human CD4+ T cells, their relative contribution to restriction of Vif-defective HIV-1 is unknown. We used HIV-1 Vif mutants that were defective in their ability to degrade either A3G or A3F/A3D and found that A3G more potently restricts HIV-1 than do A3F and A3D. It is not known whether A3G and A3F can copackage into the same virions and comutate the same HIV-1 genomes. We are determining the potential of A3G and A3F to copackage into the same virions and comutate HIV-1 genomes. It is not known whether mutations induced by A3G and A3F contribute to viral genetic variation and/or influence the frequency of retroviral recombination. We are carrying out experiments to determine whether G-to-A mutations induced by A3G and A3F contribute to viral genetic diversification and evolution. _____[Corresponds to Pathak Project 2 in the October 2011 site visit report of the HIV Drug Resistance Program]