Investigating the Role of HIV-1 Integrase in the Uncoating of the Viral Core

描述(申请人提供)：艾滋病毒-1和艾滋病已被归类为一种流行病，影响世界各地。洞察病毒在人类体内经历复制周期的机制可以产生重要的信息，从而导致药物治疗，并提供关于如何治疗感染患者的病毒的总体信息。该项目旨在研究一种病毒蛋白整合酶(IN)对病毒生命周期中的一个步骤--脱壳的影响。在融合和进入细胞后，病毒核心被释放到细胞质中，核心必须在那里脱去外壳或释放衣壳(CA)蛋白，以进行后续步骤，包括将病毒RNA逆转录为cDNA，以便IN整合到宿主染色体中。尽管已对CA突变进行了研究，而且CA的稳定性已被证明对感染性至关重要，但目前对去涂层的步骤仍知之甚少。IN在这一步骤中的具体相关性尚未得到解决。我们的实验室已经通过突变分析表征了IN在反转录中的重要性。一个突变，C130S，在IN的第130位含有半胱氨酸到丝氨酸的替代。这种病毒已被证明是非传染性的，不产生逆转录产物，尽管其他步骤是正常的，逆转录酶(RT)存在并起作用。这表明缺陷一定是在逆转录时或在逆转录之前，我们假设逆转录是在脱涂层时。本项目将检测三种病毒，包括野生型、NL-C130和NL-？in，它们含有未表达的IN。我们的目标包括：确定去涂层时IN突变的缺陷，这些缺陷如何影响核心的形态和蛋白质组成，以及潜在的机制是否包括IN和CA之间的直接或间接相互作用。我们的初步结果表明，C130S取代或缺少IN会导致CA拆解的增加。我们将通过电子显微镜检查核心的形态，并使用质谱仪以及一维和二维凝胶分析来检查蛋白质的组成。我们将通过免疫共沉淀法研究IN和CA之间可能的相互作用。该项目的结果无疑将给病毒生命周期的揭开涂层的步骤提供洞察力，到目前为止，这一步骤仍然难以捉摸。关于IN的信息以及它如何影响病毒生命周期的许多不同步骤也将是重要的，并可随后应用于药物治疗。这个项目将研究HIV-1整合酶，一种病毒蛋白，在生命周期的脱壳步骤中的作用。我们还将确定整合酶和衣壳之间的任何相互作用，衣壳是一种已知对脱壳很重要的病毒蛋白。了解更多关于HIV-1的知识将为可能的药物治疗提供有用的信息。

DESCRIPTION (provided by applicant): HIV-1 and AIDS have been categorized as a pandemic, affecting all parts of the world. Gaining insight into the mechanisms by which the virus undergoes its replication cycle in humans can yield important information leading to drug therapy and an overall informative picture on how to treat the virus in infected patients. This project aims to investigate the effect of a viral protein, integrase (IN), on a step in the viral life cycle, uncoating. Upon fusion and entry into a cell, the viral core is released into the cytoplasm where the core must uncoat or release the capsid (CA) protein for subsequent steps to occur, including reverse transcription of viral RNA into cDNA for IN to integrate into the host chromosome. Currently uncoating is a step that remains poorly understood, although CA mutations have been investigated and CA stability has been shown to be crucial for infectivity. The specific relevance of IN in this step has not been addressed. Our lab has characterized the importance of IN on reverse transcription by mutational analysis. One mutation, C130S, contains a cysteine to serine substitution at position 130 of IN. This virus has been shown to be non-infectious, yielding no reverse transcription products, although other steps were normal and reverse transcriptase (RT) was present and functional. This suggested that the defect must be either at or prior to reverse transcription, which we hypothesize is at uncoating. This project will examine three viruses, including wildtype, NL-C130S, and NL-?IN, which contains unexpressed IN. Our aims include: determine the defects of IN mutations on uncoating, how these defects influence morphology and protein composition of cores, and if the underlying mechanism includes a direct or indirect interaction between IN and CA. Our preliminary results indicate that the C130S substitution or lack of IN cause an increase in CA disassembly. We will examine core morphology by electron microscopy and examine protein composition using mass spectrometry and 1D and 2D gel analysis. We will examine a possible interaction between IN and CA by co-immunoprecipitation. The results of this project will undoubtedly give insights to the uncoating step of the viral life cycle, which thus far has remained elusive. Information about IN and how it can affect many different steps of the viral life cycle will also be important and can subsequently be applied to drug therapy. This project will examine the effects of HIV-1 integrase, a viral protein, on the uncoating step of the life cyle. We will also determine any interaction between integrase and capsid, a viral protein known to be important for uncoating. Understanding more about HIV-1 will provide useful information for possible drug therapy.