The Cell Biology of TRIM5alpha

描述(申请人提供)：这项申请将寻求更好地了解猕猴TRIM5pha蛋白(RhTRIM5pha)在限制过程中的分子和细胞生物学，从而抑制HIV-1感染的机制。我们已经确定了一些与细胞中的TRIM5α相关的细胞蛋白。我们还开发了强大的新方法来定量表征TRIM5α与这些细胞蛋白的关联。在第一个目标中，我们将研究rh TRIM5α的自我关联能力与限制逆转录病毒感染之间的联系。具体地说，我们将表征由于我们在蛋白质的Linker2区域引入的特定突变而失去了定位到细胞质小体的能力的特定rhTRIM5α变体的影响。我们将通过最近开发的一种新的、基于成像的自关联分析来衡量这一能力，从而确定该区域中调节rhTRIM5pha自关联能力的决定因素。我们将利用这些研究中产生的知识来开发具有更强的限制HIV-1感染能力的rhTRIM5Alpha变体。在第二个目标中，我们将确定蛋白p62/seecestosome1在调节TRIM5α降解中的作用。该蛋白是一种支架蛋白，介导泛素化的货物蛋白穿梭到蛋白酶体，其已知的生物学功能使其成为在TRIM5α介导的逆转录病毒限制的生物学中发挥作用的有趣候选蛋白。我们将测试P62有条件地调节不同细胞降解途径降解TRIM5α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在细胞中发生的事件。通过监测限制期间TRIM5pha细胞质体内是否存在生物相关的细胞蛋白，我们将能够对限制进行研究，我们将能够剖析和了解这一关键的生物学过程。 与公共卫生相关：灵长类动物，包括人类，拥有TRIM5Alpha等蛋白质，这种蛋白质经过数百万年的进化，可以有效地对抗病毒感染。了解TRIM5pha介导其抗病毒作用的机制，可以将这一知识用于HIV-1感染者的抗病毒治疗

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