Nuclear Localization of HIV-1 Preintegration Complexes

描述（由申请人提供）：本提案的长期目标是确定人类免疫缺陷病毒1型（HIV-1）预整合复合体（PIC）在感染细胞中的核定位机制。先前的研究发现，病毒基质、整合酶和Vpr蛋白中的氨基酸残基在非分裂细胞中特异地起核定位作用，而最近的研究发现，在分裂细胞和非分裂细胞中，由反转录和整合酶残基Val-165和Arg-166组成的中央DNA瓣中，发现了新的核定位信号。然而，本提案中描述的初步研究驳斥了这些新序列在核易位中的作用。在中心DNA瓣携带突变的病毒在各种条件下复制。虽然在原代t细胞中发现有缺陷的皮瓣功能，但没有证据表明存在核输入缺陷。虽然整合酶突变体V165A和R166A有严重缺陷，但这些病毒主要是整合酶缺陷，而不是输入缺陷。我们确定整合酶突变体属于先前描述的多效性缺陷突变体的一类，这导致了一种假设，即核输入缺陷是各种缺陷整合酶突变体共同的表型，并且与核膜易位相比，这些突变体主要是细胞内运输缺陷。因此，多效性输入缺陷整合酶突变体将用于确定HIV-1复制复合体达到染色体整合目标所必需的细胞内步骤。参与HIV-1特异性核输入的残基将在广泛的诱变筛选后确定。宿主细胞因子对细胞内运输到染色体和PIC通过完整膜易位至关重要，将使用蛋白质相互作用测定来鉴定。这些结果将确定宿主蛋白-HIV-1相互作用对PIC核输入和细胞内运输至关重要，这将为开发针对HIV-1生命周期关键步骤的新型抗病毒药物确定靶标。

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.