Discovery and Mechanism of Antiretroviral Factors

描述（由申请人提供）：现代生物体的祖先是由病毒定植的，这些病毒很少对宿主有益，通常对宿主有害。不可避免地，宿主适应限制病毒复制，并在此过程中进化出各种自主抗病毒防御机制。其中之一是TRIM5，它是一种多聚体蛋白，可识别传入的逆转录病毒衣壳，但其确切的作用机制尚不清楚。在具体目标1中，我们将开发新技术，以了解逆转录病毒颗粒在完成其生命周期的早期阶段时，各种进入的成分发生了什么。这些试验将很有可能阐明TRIM5蛋白和其他抗逆转录病毒因子发挥其抑制作用的机制。此外，这些技术可能在研究逆转录病毒生命周期的这些步骤方面有广泛的应用。在特定的目标2中，我们将获得具有TRIM5抗性的HIV-1衣壳，该衣壳将识别TRIM5靶向的HIV-1衣壳表面残基，并将成为探索TRIM5作用机制的有用工具。此外，这些衣壳将用于产生可在恒河猴中复制的类猴性HIV毒株。除了TRIM5等已知的抗逆转录病毒蛋白外，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。在特定目标3中，我们将尝试通过构建集中的、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，这些候选基因要么(i)具有已知抗逆转录病毒基因所表现出的关键特性，要么（ii）在表现出对HIV-1或SIV感染的构成性或诱导性抗性的细胞中选择性表达。这些基因文库将被排列，每个基因将被单独测试其抑制逆转录病毒复制的能力。

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.