Cellular Cofactors of Murine Leukemia Virus Integrase

描述（由申请方提供）：本提案的总体目标是阐明细胞辅因子控制鼠白血病病毒（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，并且对于不同的逆转录病毒基因有显著差异。例如，包括MLV在内的γ-逆转录病毒有利于在转录起始位点和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伴侣可能是不同的整合位点选择性的原因。然而，直到最近，只有一个例子已经被报道：透镜上皮衍生生长因子（LEDGF/p75），它作为一个双峰系链，接合HIV-1 intasomes和导航他们的活性基因。探索γ-逆转录病毒MLV整合位点选择性的分子机制的重要性通过基于MLV的人类基因治疗载体的开发来例证。在临床试验中，使用γ-逆转录病毒载体来纠正原发性免疫缺陷是有疗效的，但不良事件的发生与基于MLV的载体插入激活原癌基因有关。我们最近发现，溴结构域和额外的末端结构域（BET）蛋白（Brd 2，3，4）是MLV IN的主要细胞结合伴侣，并证明了它们在转录起始位点靶向MLV整合的重要性。本申请旨在扩展这些重要的初步发现，以更好地理解BET蛋白如何选择性地识别MLV IN并将γ-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如何选择性且高亲和力地识别BET蛋白的结构和机制基础;目标2将研究BET蛋白介导的MLV整合和选择染色质位点之间联系的拟议双峰机制。我们的实验有望产生重要的新发现，这将有利于广泛的科学界有兴趣了解逆转录病毒整合的分子机制。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，我们的研究结果将阐明MLV和HIV-1整合位点选择性之间重要的机制相似性和差异。因此，拟议的研究将导致更好地了解不同的逆转录病毒如何进化，利用不同的细胞染色质结合伴侣插入其遗传物质在选择的位点在宿主染色体。此外，我们的研究结果将导致更好地理解人类基因治疗试验期间原癌基因附近基于MLV的载体整合的分子机制，这与患者的显著不良结局有关。

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.