Genome stability maintenance factors in early HIV-1 infection

描述（由申请人提供）：HIV-1感染受到多种宿主因素的影响，包括DNA修复途径的组成部分。其中一个例子是Rad18，它是复制后DNA修复系统的成员，也是维持基因组稳定性的重要因素。Rad18与HIV-1整合酶相互作用并使其稳定。此外，Rad18对HIV-1感染具有负向影响，这种影响发生在逆转录和整合之间的一个步骤。Rad18对MLV和腺病毒的感染也有负作用，提示双链DNA是其抑制活性的最终目标。我们假设HIV-1整合酶作为一个桥梁，允许Rad18进入病毒cDNA，在那里它可能从DNA修复途径中招募其他效应物，导致cDNA降解。我们打算确定Rad18与整合酶的相互作用是否对Rad18抑制HIV-1感染负责。这将通过评估野生型病毒和缺乏整合酶的病毒在Rad18-/-和Rad18+/+细胞中cDNA合成的差异来完成。此外，我们计划绘制参与这两个分子结合的结构域和残基。与Rad18相互作用的整合酶残基的突变将产生不能与Rad18结合的蛋白质变体，因此病毒不受Rad18的抑制。相反，与野生型Rad18相比，不能与整合酶相互作用的Rad18突变体不会对HIV-1感染产生抑制作用。我们将在病毒感染的背景下评估所有突变体。此外，我们还将研究相互作用结构域自身过表达对病毒感染的影响，以评估它们是否可以通过滴定宿主蛋白（在整合酶结构域的情况下）或通过与细胞Rad18竞争（在Rad18结构域的情况下）来抑制Rad18的抑制。我们还旨在研究在HIV-1感染中，抑制Ty反转录转位的酵母菌基因组稳定因子的人类同源物的功能保护。我们将首先分析FEN-1和WRN两个基因，它们在酵母中的同源物已知会影响Ty1的活性。敲低细胞系将使用shRNA方法获得。将对这些细胞系进行HIV-1感染测试，我们将确定病毒生命周期的哪个阶段受到影响。本研究将阐明Rad18在抑制HIV-1感染中的作用，揭示调节病毒感染的新宿主因子。这一结果将为新的抗逆转录病毒疗法的发展开辟前景。7. 项目的叙述。本研究的目的是阐明DNA修复分子Rad18抑制HIV-1的机制，并利用它们在酵母和人体内的潜在保守性，鉴定出新的HIV-1细胞抑制因子。对这些蛋白质的识别和理解可能导致利用病毒不太可能有能力适应的多重抑制的能力。这不仅可以转化为我们对病毒生物学知识的宝贵扩展，而且可能发现可调节的抗逆转录病毒靶点。

DESCRIPTION (provided by applicant): HIV-1 infection is influenced by several host factors, including components of DNA repair pathways. One example is Rad18, a member of the post-replication DNA repair system and also an important element in the maintenance of genome stability. Rad18 interacts with and stabilizes HIV-1 integrase. Moreover, Rad18 has a negative influence on HIV-1 infection, an effect exerted at a step between reverse transcription and integration. Rad18 also has a negative effect on infection by MLV, and adenovirus, suggesting that double stranded DNA is the ultimate target of the inhibitory activity. We hypothesize that HIV-1 integrase serves as a bridge to allow Rad18 to access viral cDNA, where it might recruit other effectors from DNA repair pathways, leading to cDNA degradation. We propose to determine whether Rad18 interaction with integrase is responsible for the inhibition of HIV-1 infection by Rad18. This will be accomplished by assessing the difference of cDNA synthesis between wild type viruses and viruses lacking integrase, in Rad18-/- and Rad18+/+ cells. Furthermore we plan to map the domains and residues that participate in the association of the two molecules. Mutation of the integrase residues involved in the interaction with Rad18 will yield protein variants that cannot associate with Rad18, and therefore viruses that are not inhibited by Rad18. Conversely, Rad18 mutants that cannot interact with integrase will not have an inhibitory effect on HIV-1 infection, in contrast to what observed with wild-type Rad18. We will evaluate all mutants in the context of viral infection. Furthermore, we will also examine the effect of overexpression of the interacting domains by themselves on viral infection, to assess whether they can suppress the Rad18 inhibition by titration of the host protein (in the case of integrase domains) or by competition with cellular Rad18 (in the case of Rad18 domains). We also aim to investigate the functional conservation, in HIV-1 infection, of human homologues of yeast genome stability factors that inhibit Ty retrotransposition. We will initially analyze two genes, FEN-1 and WRN, whose orthologs in yeast are known to influence Ty1 activity. Knockdown cell lines will be obtained using shRNA methodology. These cell lines will be tested for HIV-1 infection, and we will characterize which phase of the viral life cycle is affected. The proposed research will elucidate the role of Rad18 in inhibiting HIV-1 infection and reveal new host factors that modulate viral infection. The results will open perspectives for new anti-retroviral therapy development. 7. Project Narrative. The goal of this proposal is to elucidate the mechanism of HIV-1 inhibition by DNA repair molecule Rad18 and identify novel cellular suppressors of HIV-1, by taking advantage of their potential conservation from yeast to man. The identification and understanding of these proteins may lead to the ability to harness multiple levels of inhibition against which viruses would unlikely have the capacity to adapt. This can then translate not only in the precious expansion of our knowledge about the viral biology but possibly in the discovery of amenable anti- retroviral targets.