Role of Chromatin Associated Host Factors in Regulation of Retroviral Replication

项目摘要 已知许多宿主细胞因子结合逆转录病毒整合酶（IN）蛋白并促进其整合。 功能协调发展的在初步研究中，我们已经确定了一套新的主机因素，结合IN蛋白。这些 宿主因子包括组蛋白伴侣、染色质重塑物和组蛋白变体。在拟议的研究中， 我们将确定这些已确定的候选宿主因子对逆转录病毒复制的影响， 逆转录病毒属，包括α-、β-、γ-逆转录病毒以及慢病毒。我们将进一步 通过分析病毒中间体来确定宿主因素是如何影响复制的， 这一步是主人的作用。我们将在存在和不存在以下物质的情况下进行整合位点定位： 候选宿主因子，以确定对人类基因组中整合靶向的影响。 前病毒基因组在进入宿主细胞核后迅速装载组蛋白。我们 假设这些宿主因子也可能由于它们的组蛋白伴侣而促进组蛋白装载 功能因此，我们还建议评估野生型与宿主中组蛋白加载到前病毒基因组上的情况 因子敲低细胞。此外，所鉴定的宿主因子包括染色质重塑物，其在以下方面发挥作用： 将核小体间隔成图案化阵列，因此我们将分析病毒上的核小体占据谱， 基因组作为宿主因子丰度的函数。最后，IN蛋白似乎结合H2 A组蛋白变体， 因此我们将确定这些组蛋白变体是否被募集并沉积在前病毒基因组上。 我们将通过敲除研究进一步确定组蛋白变体是否影响病毒复制。我们预计 这些研究将揭示逆转录病毒复制的新方面，因此确定了逆转录病毒的全新靶点。 干预 !

Project Summary A number of host cell factors are known to bind the retroviral integrase (IN) protein and facilitate its functions. In preliminary studies we have identified a novel set of host factors that bind the IN protein. These host factors include histone chaperones, chromatin remodelers and histone variants. In the proposed research, we will determine the effect of these identified candidate host factors on retroviral replication, testing multiple genera of retroviruses including alpha-, beta-, gammaretroviruses as well as lentiviruses. We will further determine how the host factor is affecting replication by analyzing viral intermediates to uncover the specific step at which the host factor acts. We will perform integration site mapping in the presence and absence of candidate host factors to determine the effects on integration targeting in the human genome. The proviral genome is rapidly loaded with histones upon entry into the host cell nucleus. We hypothesize that these host factors may also be facilitating histone loading due to their histone chaperone function. Therefore, we also propose to evaluate histone loading onto the proviral genome in wild type vs. host factor knockdown cells. Further, the identified host factors include chromatin remodelers that play a role in spacing nucleosomes into patterned arrays and thus we will analyze nucleosome occupancy profile on the viral genome as a function of host factor abundance. Lastly, the IN protein appears to bind H2A histone variants, and thus we will determine whether these histone variants are recruited and deposited on the proviral genome. We will further determine if histone variants affect viral replication through knockout studies. We expect that these studies will reveal new aspects of retrovirus replication and therefore define entirely new targets for intervention. !