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

描述（由申请人提供）：HIV-1和艾滋病已被列为流行病，影响世界各地。深入了解病毒在人体内复制周期的机制可以产生重要的信息，从而导致药物治疗和如何治疗感染患者中的病毒的整体信息。该项目旨在研究病毒蛋白整合酶（IN）对病毒生命周期中的一个步骤（脱壳）的影响。在融合并进入细胞后，病毒核心释放到细胞质中，在细胞质中核心必须脱壳或释放衣壳（CA）蛋白以进行后续步骤，包括将病毒RNA逆转录成cDNA以使IN整合到宿主染色体中。目前，去包被是一个仍然知之甚少的步骤，尽管已经研究了CA突变，并且CA稳定性已被证明对感染性至关重要。IN在此步骤中的具体相关性尚未得到解决。本实验室通过突变分析研究了IN在逆转录中的重要性。一个突变C130 S在IN的130位含有半胱氨酸到丝氨酸的取代。这种病毒已被证明是非感染性的，不产生逆转录产物，尽管其他步骤是正常的，逆转录酶（RT）是存在和功能。这表明，缺陷必须在逆转录或之前，我们假设是在脱壳。该项目将研究三种病毒，包括野生型，NL-C130 S，和NL-？IN，其中包含未表示的IN。我们的目标包括：确定IN突变在脱壳时的缺陷，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，C130 S取代或缺乏IN导致CA拆卸增加。我们将通过电子显微镜检查核心形态，并使用质谱和一维和二维凝胶分析检查蛋白质组成。我们将通过免疫共沉淀研究IN和CA之间可能的相互作用。该项目的结果无疑将为病毒生命周期的脱壳步骤提供见解，迄今为止，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，并且可以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）对生命周期中去包被步骤的影响。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的信息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.