Host factors impact in early phases of HIV-1 infection

描述(由申请人提供)：将HIV-1基因组强制插入宿主染色体DNA是逆转录病毒区别于其他病毒的特征之一。在细胞核中，病毒和细胞因子相互作用，以确保病毒DNA正确整合到宿主染色质中。此外，修复插入部位是实现生产性感染的根本。在细胞配备的几种DNA修复途径中，有一些已经在体外和体内进行了测试，以确定它们是否有能力促进整合缺口修复。我们已经证明整合酶与两个不同的细胞过程相互作用，一个是蛋白酶体降解途径(N-端规则)，另一个是DNA修复途径(复制后修复/跨损伤)。这两条途径共享关键分子Rad6的活性。我们已经通过整合酶的突变表明，决定其N端规则依赖稳定性的元件在HIV-1感染中具有重要意义。这项提议的目的是调查这两条途径在多大程度上影响艾滋病毒-1感染的早期步骤，并特别强调整合。最初，我们将确定病毒生命周期早期阶段的哪个阶段受到整合酶第一位点突变的抑制，因为该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背景下观察到的表型的贡献。DNA修复对于HIV-1感染至关重要。迄今为止，除了我们观察到其主要成分hRadl 8与整合酶hRadl 8之间存在直接相互作用外，复制后DNA修复途径尚未与逆转录病毒整合有关，hRadl 8也与hRad6结合。HRadl 8在HIV-1复制中的作用将通过感染过度表达这种蛋白质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式反应确定，并将评估对插入连接处实际DNA缺口修复的影响。这项建议中描述的实验结果有望为我们提供一组关于这两个相互交叉的细胞通路对HIV-1复制的影响的信息。此外，我们还将了解干扰Nend规则和复制后DNA修复/翻译是否最终能够使宿主细胞对感染产生抵抗力，从而使这两条途径有资格成为抗逆转录病毒治疗的潜在靶点。

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.