Genome stability maintenance factors in early HIV-1 infection

描述（由申请人提供）：HIV-1 感染受多种宿主因素影响，包括 DNA 修复途径的组成部分。 Rad18就是一个例子，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元件。 Rad18 与 HIV-1 整合酶相互作用并稳定 HIV-1 整合酶。此外，Rad18 对 HIV-1 感染具有负面影响，这种影响是在逆转录和整合之间的步骤中产生的。 Rad18 对 MLV 和腺病毒的感染也有负面影响，表明双链 DNA 是抑制活性的最终目标。我们假设 HIV-1 整合酶充当桥梁，允许 Rad18 访问病毒 cDNA，它可能会从 DNA 修复途径中招募其他效应子，导致 cDNA 降解。我们建议确定 Rad18 与整合酶的相互作用是否是 Rad18 抑制 HIV-1 感染的原因。这将通过评估 Rad18-/- 和 Rad18/ 细胞中野生型病毒和缺乏整合酶的病毒之间 cDNA 合成的差异来完成。此外，我们计划绘制参与两个分子关联的结构域和残基。参与与 Rad18 相互作用的整合酶残基的突变将产生无法与 Rad18 结合的蛋白质变体，因此病毒不会被 Rad18 抑制。相反，与野生型 Rad18 观察到的情况相反，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，我们还将检查相互作用结构域本身的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 抑制。我们还旨在研究抑制 Ty 逆转录转座的酵母基因组稳定因子的人类同源物在 HIV-1 感染中的功能保守性。我们将首先分析两个基因：FEN-1 和 WRN，已知它们在酵母中的直系同源物会影响 Ty1 活性。将使用 shRNA 方法获得敲低细胞系。这些细胞系将接受 HIV-1 感染检测，我们将确定病毒生命周期的哪个阶段受到影响。拟议的研究将阐明 Rad18 在抑制 HIV-1 感染中的作用，并揭示调节病毒感染的新宿主因素。研究结果将为新的抗逆转录病毒疗法的开发开辟前景。 7. 项目叙述。该提案的目标是阐明 DNA 修复分子 Rad18 抑制 HIV-1 的机制，并利用其从酵母到人类的潜在保守性，鉴定新型 HIV-1 细胞抑制因子。对这些蛋白质的识别和理解可能会导致利用多种抑制水平的能力，而病毒不太可能有能力适应这些抑制。这不仅可以转化为我们对病毒生物学知识的宝贵扩展，而且可能转化为适合的抗逆转录病毒靶标的发现。

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.