Inhibition of Integrase-Mediated Viral nuclear Transport

描述(由申请人提供)：艾滋病毒-1的核进口是病毒生命周期中的关键步骤，迄今尚未被用作抗逆转录病毒治疗的目标。因此，我们在HIV-1整合酶蛋白的羧基末端发现了不连续的碱性氨基酸簇，它们共同作用，在天然整合酶蛋白和原代细胞培养感染的背景下影响强大的核定位信号(HIV-1 IN NLS)。事实上，我们对包含这些突变的HIV-1分子克隆的分析表明，HIV-1IN NLS是病毒在几种已知支持HIV感染的细胞类型中复制的绝对要求，包括人巨噬细胞和激活的外周血单核细胞。我们的突变体在分裂和非潜水细胞方面存在缺陷的事实与最近的数据一致，这些数据表明HIV-1基因组的核运输独立于周期细胞中有丝分裂的核分解(Katz等人，J.Virol。77：13412-13417)。尽管协调整合酶介导的病毒核运输所需的细胞因子在很大程度上仍不清楚，但最近提出了几个可能对这一过程做出贡献的候选因子。例如，EED(胚胎外胚层发育)是一种多梳状蛋白质，与核孔复合体附近的整合酶共定位，并在与我们已确定具有NLS活性的区域重叠的区域结合整合酶(Violot等人，J.Virol。77：12507-22)。有趣的是，整合酶和EED之间的特异性结合可以与展示多肽片段的噬菌体竞争，这些片段映射到我们已经确定的体内整合酶NLS功能关键的一组带电簇氨基酸残基。在这项提议中，我们试图澄清EED和其他尚未确定的细胞因素对HIV-1整合酶介导的核运输的贡献。我们描述的整合酶NLS突变体将是这些分析的关键工具。这些研究反过来将为开发检测艾滋病毒-1整合酶和必要的细胞辅因子(S)之间的相互作用提供理论基础，并有可能被抑制。我们假设，阻断整合酶与其相关细胞伙伴之间的相互作用将导致整合前一步病毒生命周期的废除。因此，HIV-1整合酶介导的核输入的小分子(或类似肽)抑制剂将代表一类新的抗病毒药物来遏制HIV感染。

DESCRIPTION (provided by applicant): Nuclear import of HIV-1 is a key step in the viral life cycle that to date has not been exploited as a target for anti-retroviral therapy. Accordingly, we have identified discontinuous charged-clusters of basic amino acids in the carboxyl-terminus of the HIV-1 integrase protein that act together to affect a powerful nuclear localization signal (HIV-1 IN NLS) in the context of both the native integrase protein and in infection of primary cell culture. Indeed, our analysis of HIV-1 molecular clones containing these mutations demonstrates that the HIV-1 IN NLS is an absolute requirement for viral replication in several cell types known to support HIV infection including human macrophages and activated peripheral blood mononuclear cells. The fact that our mutants are defective in dividing as well as non-diving cells is concordant with recent data indicating that nuclear transport of the HIV-1 genome is independent of mitotic nuclear disassembly in cycling cells (Katz et al., J. Virol. 77: 13412-13417). Although the requisite cellular factors that coordinate integrase-mediated viral nuclear transport remain largely unknown, several candidate factors have recently been put forward as likely contributors to this process. For example, EED (embryonic ectoderm development), a polycomb-group protein, co-localizes with integrase in the vicinity of the nuclear pore complex and binds integrase in a region that overlaps with the domain we have identified as having NLS activity (Violot et al., J. Virol. 77:12507-22). Intriguingly, specific binding between integrase and EED can be competed with phage that display peptide segments that map to the same set of charged-cluster amino acid residues that we have identified as critical to integrase NLS function in vivo. In this proposal we seek to clarify the contribution made by EED, and other cellular factors that have yet to be identified to HIV-1 integrase-mediated nuclear transport. The integrase NLS mutants we have described will be critical tools for these analyses. These studies, in turn will provide the rationale for development of assays in which the interaction between HIV-1 integrase and the requisite cellular co-factor(s) can be examined and potentially inhibited. We hypothesize that blockade of the interactions between integrase and its relevant cellular partners will result in the abrogation of the viral life cycle at a step preceding integration. Therefore, small molecule (or peptido-mimetic) inhibitors of HIV-1 integrase-mediated nuclear import would represent a new class of antiviral agents to curtail HIV infection.