Cellular Cofactors of Murine Leukemia Virus Integrase

描述(由申请人提供)：本提案的主要目标是阐明细胞辅因子控制小鼠白血病病毒(MLV)整合位点在染色质中分布的分子机制。逆转录病毒整合的染色体靶点的选择不是随机的，不同的逆转录病毒基因有明显的差异。例如，包括MLV在内的伽玛逆转录病毒倾向于在转录起始点和CpG岛附近整合，而包括HIV-1在内的慢病毒则优先整合在活性基因内。这些观察结果表明，逆转录病毒整合酶的不同细胞结合伙伴可能对不同的整合位点选择性负责。然而，直到最近，只有一个例子被报道：晶状体上皮源性生长因子(LEDGF/p75)，它的功能是作为一条双峰纽带，与HIV-1内含体接触，并将它们导航到活性基因。以MLV为基础的人类基因治疗载体的发展证明了探索伽马-逆转录病毒整合位点选择性的分子机制的重要性。在临床试验中，使用伽马逆转录病毒载体来纠正原发免疫缺陷已经是治愈的，但也出现了与MLV载体插入激活原癌基因相关的不良事件。我们最近发现，溴结构域和额外末端结构域(BET)蛋白(Brd2，3，4)是MLV IN的主要细胞结合伙伴，并证明了它们在转录起始点靶向整合MLV的重要性。本申请旨在扩大这些重要的初步发现，以更好地了解BET蛋白如何选择性识别MLV IN并将伽马-逆转录病毒整合导航到染色质中的特定位点的潜在机制。特别是，目标1将研究MLV IN如何选择性和高亲和力识别BET蛋白的结构和机制基础；目标2将研究拟议的BET蛋白介导的MLV整合和特定染色质位点之间的联系的双峰机制。我们的实验有望产生重要的新发现，这将使对了解逆转录病毒整合的分子机制感兴趣的广泛科学界受益。由于BET蛋白-MLV IN相互作用只是第二个系统，除了HIV-1 IN-LEDGF/p75相互作用外，我们的发现将阐明MLV和HIV-1整合位点选择性之间的重要机制的异同。因此，拟议的研究将有助于更好地理解不同的逆转录病毒是如何进化到利用不同的细胞染色质结合伙伴在宿主染色体的选择位点插入它们的遗传物质的。此外，我们的发现将有助于更好地理解在人类基因治疗试验中整合原癌基因附近基于MLV的载体的分子机制，这与患者的重大不良后果有关。

DESCRIPTION (provided by applicant): The overarching goal of the present proposal is to elucidate the molecular mechanisms by which cellular cofactors control the distribution of murine leukemia virus (MLV) integration sites in chromatin. The selection of chromosomal targets for retroviral integration is not random and varies markedly for different retroviral gener. For example, the gamma-retroviruses including MLV favor integration near transcription start sites and CpG islands, whereas lentiviruses including HIV-1 preferentially integrate within active genes. These observations have suggested that different cellular binding partners of retroviral integrases could be responsible for distinct integration site selectivity. However, until very recently, only one example has been reported: lens epithelium-derived growth factor (LEDGF/p75), which functions as a bimodal tether that engages HIV-1 intasomes and navigates them to active genes. The significance of exploring the molecular mechanisms of gamma-retroviral MLV integration site selectivity is exemplified by the development of MLV-based vectors for human gene-therapy. In clinical trials, the use of gamma-retroviral vectors to correct primary immunodeficiencies has been curative, but adverse events have occurred associated with insertional activation of protooncogenes by MLV-based vectors. We have recently discovered that the bromodomain and extra terminal domain (BET) proteins (Brd2, 3, 4) are the principal cellular binding partners of MLV IN and demonstrated their significance for targeting MLV integration at transcription start sites. The present application aims to extend these important initial findings to better understand the underlying mechanism for how BET proteins selectively recognize MLV IN and navigate the gamma-retroviral integration to specific sites in chromatin. In particular, aim 1 will study structural and mechanistic foundations for how MLV IN recognizes BET proteins selectively and with high affinity; and aim 2 will examine a proposed bimodal mechanism for BET proteins-mediated link between MLV integration and select chromatin sites. Our experiments are expected to yield important novel findings, which will benefit a wide scientific community interested in understanding molecular mechanisms of retroviral integration. Since BET proteins-MLV IN interactions are only the second system, besides HIV-1 IN-LEDGF/p75 interactions, our findings will elucidate important mechanistic similarities and differences between MLV and HIV-1 integration site selectivity. Thus, the proposed studies will lead to better understanding of how different retroviruses have evolved to utilize distinct cellular chromatin binding partners to insert their genetic material at select sits in the host chromosome. Additionally, our findings will lead to better understanding of molecular mechanisms for integrations of MLV-based vectors near proto-oncogenes during human gene-therapy trails, which have been linked to significant adverse outcomes in patients.