Nuclear Localization of HIV-1 Preintegration Complexes

描述(申请人提供)：这项建议的长期目标是确定人类免疫缺陷病毒1型(HIV-1)整合前复合体(PIC)在感染细胞中的核定位的机制。以前的工作发现了病毒基质中的氨基酸残基，整合酶和VPR蛋白在未分裂细胞中具有核定位功能，最近的发现发现在中央DNA瓣中由逆转录和整合酶残基Val-165和Arg-166产生的新的核定位信号，这些残基在分裂和未分裂细胞中都起作用。然而，该提案中描述的初步研究驳斥了这些新序列在核转位中的作用。病毒在中央DNA翻盖中携带突变，在各种条件下复制。虽然在原始T细胞中发现了瓣功能缺陷，但没有证据表明核进口缺陷。虽然整合酶突变体V165A和R166A是严重缺陷的，但这些病毒主要是整合酶缺陷的，而不是输入缺陷的。整合酶突变体被确定为先前描述的多向性缺陷突变的一类，从而导致假设缺陷核输入是多种缺陷整合酶突变的共同表型，并且与核膜转位相比，这些突变主要是细胞内转运缺陷。因此，多效性输入缺陷整合酶突变体将被用来确定HIV-1复制复合体达到整合的染色体目标所必需的细胞内步骤。在广泛的诱变筛选后，将确定HIV-1 PIC的特定核进口中涉及的残留物。对于细胞内转运到染色体和PIC通过完整的膜转运至关重要的宿主细胞因子将使用蛋白质-蛋白质相互作用分析来鉴定。结果将确定宿主蛋白-HIV-1相互作用对于PIC核进口和细胞内贩运至关重要，这将确定针对HIV-1生命周期关键步骤开发新型抗病毒药物的目标。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to identify the mechanism of human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC) nuclear localization in infected cells. Previous work identified amino acid residues in the viral matrix, integrase and Vpr proteins that functioned in nuclear localization specifically in nondividing cells, and more recent findings identified novel nuclear localization signals in the central DNA flap made by reverse transcription and integrase residues Val-165 and Arg-166 that functioned in both dividing and nondividing cells. However the Preliminary Studies described in this proposal refute the roles of these novel sequences in nuclear translocation. Viruses carrying mutations in the central DNA flap replicated under a variety of conditions. Although defective flap function was identified in primary T-cells, there was no evidence for a nuclear import defect. Although integrase mutants V165A and R166A were acutely defective, these viruses were primarily integrase-defective, not import defective. It was determined that the integrase mutants fell into a category of previously-described pleiotropically defective mutants, leading to the hypothesis that defective nuclear import is a phenotype common to a variety of defective integrase mutants, and that these mutants are primarily defective for intracellular trafficking as compared to nuclear membrane translocation. Thus, pleiotropic import-defective integrase mutants will be used to determine intracellular steps essential for HIV-1 replication complexes to reach the chromosomal targets of integration. Residues involved in specific nuclear import of HIV-1 PICs will be identified following extensive mutagenesis screens. Host cell factors essential for intracellular trafficking to chromosomes and PIC translocation through intact membranes will be identified using protein-protein interaction assays. The results will determine host protein-HIV-1 interactions essential for PIC nuclear import and intracellular trafficking, which should define targets for the development of novel antiviral drugs against essential steps in the HIV-1 life cycle.