The Cell Biology of TRIM5alpha

描述（由申请人提供）：本申请将通过定义限制期间rhTRIM5alpha的分子和细胞生物学来更好地了解恒河猴TRIM5alpha蛋白（rhTRIM5alpha）抑制HIV-1感染的机制。我们已经在细胞中发现了许多与trim5 α相关的细胞蛋白。我们还开发了强大的新方法来定量表征TRIM5alpha与这些细胞蛋白的关联。在第一个目标中，我们将研究rhTRIM5alpha自我结合能力和限制逆转录病毒感染之间的联系。具体来说，我们将描述特异性rhtrim5 α变异体的影响，这些变异体由于我们在蛋白质的连接子2区域引入了特异性突变而失去了定位到细胞质体的能力。我们将确定该区域中介导rhTRIM5alpha自我关联能力的决定因素，并使用我们最近开发的一种新颖的、基于成像的自我关联试验来测量这种能力。我们将利用这些研究中产生的知识来开发具有更强限制HIV-1感染能力的rhTRIM5alpha变体。在第二个目标中，我们将确定p62/sequestosome1蛋白在调节trim5 α降解中的作用。该蛋白是一种介导泛素化货物蛋白向蛋白酶体穿梭的支架蛋白，已知的生物学功能使其成为TRIM5alpha介导的逆转录病毒限制生物学中发挥作用的有趣候选者。我们将检验p62有条件地调节不同细胞降解途径降解TRIM5alpha的能力的假设。在第三个目标中，我们将使用定量成像方法来定义当限制性敏感病毒粒子进入靶细胞的细胞质时细胞内发生的事件。通过在限制期间监测trim5 α细胞质体中生物相关细胞蛋白的存在，我们将能够进行限制，我们将能够解剖并了解这一关键的生物过程。

DESCRIPTION (provided by applicant): This application will seek to better understand the mechanism by which the TRIM5alpha protein from rhesus macaques (rhTRIM5alpha) inhibits HIV-1 infection by defining the molecular and cellular biology of rhTRIM5alpha during restriction. We have identified a number of cellular proteins that associate with TRIM5alpha in cells. We have also developed powerful new methods to quantitatively characterize the association of TRIM5alpha with these cellular proteins. In the first aim, we will examine the connection between the ability of rhTRIM5alpha to self-associate and restrict retroviral infection. Specifically, we will characterize the effects of specific rhTRIM5alpha variants that have lost the ability to localize to cytoplasmic bodies due to specific mutations we have introduced into the linker2 region of the protein. We will identify the determinants in this region that mediate ability of rhTRIM5alpha to self-associate, measuring this ability with a novel, imaging based self-association assay we have recently developed. We will utilize the knowledge generated in these studies to develop rhTRIM5alpha variants with increased ability to restrict HIV-1 infection. In the second aim, we will define the role of the protein p62/sequestosome1 in regulating the degradation of TRIM5alpha. The known biological function of this protein, a scaffolding protein that mediates the shuttling of ubiquitylated cargo proteins to the proteasome, makes it an interesting candidate to play a role in the biology of TRIM5alpha mediated retroviral restriction. We will test the hypothesis that p62 conditionally regulates the ability of different cellular degradative pathways to degrade TRIM5alpha. In the third aim, we will use quantitative imaging methods to define the events that occur in cells when restriction sensitive virions enter the cytoplasm of target cells. By monitoring the presence of biologically relevant cellular proteins in TRIM5alpha cytoplasmic bodies during restriction, we will be able g restriction, we will be able to dissect and develop an understanding of this critical biological process. PUBLIC HEALTH RELEVANCE: Primates, including humans, possess proteins, such as TRIM5alpha that have evolved for millions of years to effectively combat viral infection. Understanding the mechanisms by which TRIM5alpha mediates its antiviral effects could allow this knowledge to be harnessed in the form of antiviral therapy for people infected with HIV-1