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rhTRIM 5 α的分子和细胞生物学，寻求更好地理解来自恒河猴的TRIM 5 α蛋白（rhTRIM 5 α）抑制HIV-1感染的机制。我们已经鉴定了许多与细胞中TRIM 5 α相关的细胞蛋白。我们还开发了强有力的新方法来定量表征TRIM 5 α与这些细胞蛋白的关联。在第一个目标中，我们将研究rhTRIM 5 α自我关联和限制逆转录病毒感染的能力之间的联系。具体来说，我们将表征特定rhTRIM 5 α变体的影响，这些变体由于我们引入蛋白质的连接子2区域的特定突变而失去了定位于胞质体的能力。我们将确定该区域中介导rhTRIM 5 α自我关联能力的决定因素，并使用我们最近开发的一种新的基于成像的自我关联测定来测量这种能力。我们将利用这些研究中产生的知识开发rhTRIM 5alpha变体，以提高限制HIV-1感染的能力。在第二个目标中，我们将定义蛋白p62/螯合体1在调节TRIM 5 α降解中的作用。这种蛋白的已知生物学功能，介导泛素化货物蛋白穿梭到蛋白酶体的支架蛋白，使其成为在TRIM 5 α介导的逆转录病毒限制的生物学中发挥作用的有趣候选者。我们将测试p62有条件地调节不同细胞降解途径降解TRIM 5 alpha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敏感病毒体进入靶细胞的细胞质时在细胞中发生的事件。通过在限制性内切酶过程中监测TRIM 5 α胞质体中生物学相关细胞蛋白的存在，我们将能够对限制性内切酶进行分析，并对这一关键的生物学过程进行理解。 公共卫生相关性：包括人类在内的灵长类动物拥有TRIM 5 α等蛋白质，这些蛋白质已经进化了数百万年，可以有效地对抗病毒感染。了解TRIM 5 α介导其抗病毒作用的机制可以使这一知识以抗病毒治疗的形式用于HIV-1感染者

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