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Host factors impact in early phases of HIV-1 infection

宿主因素对 HIV-1 感染早期阶段的影响

基本信息

批准号：
6853571
负责人：
LUBBERTUS C MULDER
金额：
$ 25.65万
依托单位：
AARON DIAMOND AIDS RESEARCH CENTER
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-02-15 至 2007-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6853571
关键词：
DNA repair flow cytometry host organism interaction human immunodeficiency virus 1 integrase polymerase chain reaction protein structure function proteolysis virus infection mechanism virus replication

项目摘要

DESCRIPTION (provided by applicant): The obligated insertion of the HIV-1 genome into the host chromosomal DNA is one of the features that differentiates retroviruses from other viruses. In the nucleus, viral and cellular factors cooperate in order to ensure the correct integration of viral DNA in the host chromatin. Moreover repair of the insertion site is fundamental in order to achieve a productive infection. Among the several DNA repair pathways the cell is equipped with, some have been tested both in vitro as well as in vivo for their ability to promote integration gap repair. We have demonstrated the interaction of integrase with two different cellular processes, a proteasome degradation pathway (N-end rule) and a DNA repair pathway (post-replication repair/translesion). These two pathways share the activity of the key molecule, Rad6. We have shown by mutagenesis of integrase, that elements that determine its N-end rule dependent stability, are significant in HIV-1 infection. The aim of this proposal is to investigate to what extent the two pathways influence the early steps of HIV-1infection with special emphasis dedicated to integration. Initially we will determine which phase within the early steps of viral life cycle is inhibited by mutations in the first position of integrase, since this residue is key to its degradation. Furthermore we will analyze the contribution of Rad6 to both integrase stability as well as to the phenotype observed in the context of the viral mutants. DNA repair is essential for HIV-1infection. To date the post-replication DNA repair pathway has not yet been implicated in retroviral integration other than by our observation that there is a direct interaction between one of its principal components, hRadl 8, and integrase, hRadl 8 also binds hRad6. The role hRadl 8 in HIV-1 replication will be studied by infection of cells overexpressing this protein or molecules that inhibit its endogenous expression. The steps mostly affected by these treatments will be identified by real-time PCR, and the effect on the actual DNA gap repair at the insertion junctions will be assessed as well. The results of the experiments described in this proposal are expected to provide us with a set of information about the influence of these two intercrossing cellular pathways on HIV-1 replication. Moreover we will learn whether interfering with the Nend rule and post-replication DNA repair/translation can eventually render the host cell resistant to infection, and thus qualify the two pathways as potential targets for anti-retroviral therapy.

描述（由申请人提供）：HIV-1基因组强制插入宿主染色体DNA是将逆转录病毒与其他病毒区分开来的特征之一。在细胞核内，病毒和细胞因子相互配合，以确保病毒DNA在宿主染色质上的正确整合。此外，修复插入部位是实现生产性感染的基础。在细胞配备的几种DNA修复途径中，一些已经在体外和体内测试了它们促进整合间隙修复的能力。我们已经证明了整合酶与两种不同的细胞过程的相互作用，蛋白酶体降解途径（n端规则）和DNA修复途径（复制后修复/翻译）。这两种途径共享关键分子Rad6的活性。我们已经通过整合酶的诱变表明，决定其n端规则依赖稳定性的元件在HIV-1感染中是重要的。本提案的目的是研究这两种途径在多大程度上影响hiv -1感染的早期步骤，并特别强调整合。首先，我们将确定在病毒生命周期的早期阶段哪个阶段被整合酶第一个位置的突变所抑制，因为这个残基是其降解的关键。此外，我们将分析Rad6对整合酶稳定性以及在病毒突变体中观察到的表型的贡献。DNA修复对hiv -1感染至关重要。迄今为止，复制后DNA修复途径尚未涉及逆转录病毒整合，除了我们观察到其主要成分之一hRadl 8与整合酶之间存在直接相互作用，hRadl 8也结合hRad6。hRadl 8在HIV-1复制中的作用将通过感染过表达该蛋白的细胞或抑制其内源性表达的分子来研究。受这些处理影响最大的步骤将通过实时聚合酶链反应（real-time PCR）确定，对插入连接处实际DNA间隙修复的影响也将被评估。本提案中描述的实验结果有望为我们提供一组关于这两种交叉细胞通路对HIV-1复制的影响的信息。此外，我们将了解干扰Nend规则和复制后DNA修复/翻译是否最终能使宿主细胞抵抗感染，从而使这两种途径成为抗逆转录病毒治疗的潜在靶点。