Inhibition of Integrase-Mediated Viral Nuclear Transport

描述（由申请人提供）：HIV-1的核输入是病毒生命周期中的关键步骤，迄今为止尚未被用作抗逆转录病毒治疗的靶点。因此，我们已经确定了在HIV-1整合酶蛋白的羧基末端的碱性氨基酸的不连续带电簇，其在天然整合酶蛋白和原代细胞培养物感染的背景下一起作用以影响强大的核定位信号（HIV-1 IN NLS）。事实上，我们对含有这些突变的HIV-1分子克隆的分析表明，HIV-1 IN NLS是已知支持HIV感染的几种细胞类型（包括人巨噬细胞和活化的外周血单核细胞）中病毒复制的绝对要求。我们的突变体在分裂和非分裂细胞中有缺陷的事实与最近的数据一致，该数据表明HIV-1基因组的核转运不依赖于循环细胞中的有丝分裂核解体（Katz等人，J. Virol. 77：13412-13417）。虽然协调整合酶介导的病毒核转运所需的细胞因子在很大程度上仍然未知，但最近提出了几个候选因子作为这一过程的可能贡献者。例如，EED（胚胎外胚层发育），一种多梳组蛋白，与整合酶共定位在核孔复合物附近，并在与我们已鉴定为具有NLS活性的结构域重叠的区域中结合整合酶（Violot et al. 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噬菌体竞争，所述噬菌体展示映射到我们已经鉴定为对体内整合酶NLS功能至关重要的同一组带电簇氨基酸残基的肽段。在这个建议中，我们试图澄清EED和其他尚未确定的细胞因子对HIV-1整合酶介导的核转运的贡献。我们所描述的整合酶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方法提供理论依据，在该方法中，可以检查并潜在抑制HIV-1整合酶与必要的细胞辅因子之间的相互作用。我们假设，整合酶及其相关细胞伴侣之间的相互作用的阻断将导致在整合前的一个步骤废除病毒的生命周期。因此，HIV-1整合酶介导的核输入的小分子（或肽模拟物）抑制剂将代表一类新的抗病毒药物，以减少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.