Discovery and Mechanism of Antiretroviral Factors

描述(申请人提供)：现代生物体的祖先被极少有益的病毒定居，而这些病毒往往对宿主有害。不可避免地，宿主适应了限制病毒复制，并在这样做的过程中进化出了各种自主的抗病毒防御机制。其中之一是TRIM5，它是一种识别进入的逆转录病毒衣壳的多聚体蛋白，但其确切的作用机制尚不清楚。在具体目标1中，我们将开发新的技术，以了解逆转录病毒颗粒的各种传入成分在完成其生命周期的早期步骤时会发生什么。这些检测将很可能阐明TRIM5蛋白和其他抗逆转录病毒因子发挥抑制作用的机制。此外，这些技术很可能在研究逆转录病毒生命周期的这些步骤中有广泛的应用。在具体目标2中，我们将获得抗TRIM5的HIV-1衣壳，它将识别TRIM5靶向的HIV-1衣壳表面的残基，并将为探索TRIM5的作用机制提供有用的工具。此外，这些衣壳将被用来产生可以在恒河猴身上复制的嗜猴艾滋病毒毒株。除了像TRIM5这样的已知抗逆转录病毒蛋白外，还有几个理由认为还有更多，也许更多的抗病毒宿主防御基因有待发现。在具体目标3中，我们将试图通过构建集中排列的cDNA文库来鉴定这些基因，每个文库由数十到数百个候选基因组成，这些文库或者(I)具有已知抗逆转录病毒基因所表现出的关键特性，或者(Ii)选择性地在对HIV-1或SIV感染表现出结构性或诱导性抗性的细胞中表达。这些基因库将被排列，每个基因将被单独测试其抑制逆转录病毒复制的能力。 与公共卫生相关：人类和实验动物有一系列的抗病毒防御机制，我们对这些机制只有一个初步的了解。识别和了解抗逆转录病毒基因产品的作用机制可能会导致治疗包括艾滋病在内的传染病的全新化疗策略。此外，了解特定物种的变异和对抗逆转录病毒基因的工程抗性有助于发展人类逆转录病毒感染的动物模型。

DESCRIPTION (provided by applicant): The ancestors of modern organisms were colonized by viruses that were very rarely beneficial, and often deleterious to the host. Inevitably, hosts adapted to limit viral replication, and in so doing evolved various autonomous antiviral defense mechanisms. One of these is TRIM5, which is multimeric protein that recognizes incoming retroviral capsids, but its precise mechanisms of action are unclear. In specific aim 1 we will develop new techniques to understand what happens to various incoming components of retroviral particles as they complete the early steps of their life cycle. These assays will very likely illuminate the mechanisms by which TRIM5 proteins and other antiretroviral factors exert their inhibitory effects. Moreover, these techniques are likely to have a wide range of applications in the study of these steps of the retroviral life cycle. In specific aim 2 we will derive TRIM5-resistant HIV-1 capsids that will identify residues on the surface of the HIV-1 capsid that are targeted by TRIM5 and will serve a useful tools to probe the mechanism of action of TRIM5. Additionally these capsids will be used for generating simian-tropic HIV- strains that can replicate in rhesus macaques. In addition to known antiretroviral proteins like TRIM5, there are several reasons to think that there are more, perhaps many more, antiviral host defense genes in to be discovered. In specific aim 3, we will attempt to identify such genes by constructing focused, arrayed cDNA libraries, each consisting of tens to hundreds of candidate genes, that either (i) have key properties exhibited by known antiretroviral genes or (ii) are selectively expressed in cells that exhibit constitutive or induced resistance to HIV-1 or SIV infection. These genes libraries will be arrayed each gene will be individually tested for its ability to inhibit retrovirus replication. PUBLIC HEALTH RELEVANCE: Humans and laboratory animals have an array of antiviral defense mechanisms, of which we have only a rudimentary understanding. Identifying and understanding the mechanism of action of antiretroviral gene products could lead to completely new chemotherapeutic strategies for tackling infectious diseases, including AIDS. In addition, understanding species-specific variation and engineering resistance to antiretroviral genes could facilitate the development of animal models of human retroviral infection.