Nuclear Localization of HIV-1 Preintegration Complexes

超出所提供的空间。该提案的长期目标是确定人类免疫缺陷病毒1型（HIV-1）整合前复合物（PIC）在感染细胞中的核定位机制。以前的工作确定了病毒基质中的氨基酸残基，整合酶和Vpr蛋白，在核定位功能，特别是在nondividing细胞，和最近的研究结果确定了新的核定位信号在中央DNA瓣由逆转录和整合酶残基瓦尔-165和Arg-166，在分裂和nondividing细胞。然而，该提案中描述的初步研究反驳了这些新序列在核转位中的作用。病毒携带突变的中央DNA瓣复制在各种条件下。虽然在原代T细胞中发现了有缺陷的皮瓣功能，但没有证据表明核输入缺陷。虽然整合酶突变体V165 A和R166 A是急性缺陷型，但这些病毒主要是整合酶缺陷型，而不是输入缺陷型。确定整合酶突变体属于先前描述的多效性缺陷突变体的类别，从而导致以下假设：缺陷性核输入是多种缺陷性整合酶突变体共有的表型，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复制复合物到达整合的染色体靶点所必需的细胞内步骤。在广泛的诱变筛选后，将鉴定参与HIV-1 PIC特异性核输入的残基。将使用蛋白质-蛋白质相互作用试验鉴定细胞内转运至染色体和PIC通过完整膜易位所必需的宿主细胞因子。这些结果将确定PIC核输入和细胞内运输所必需的宿主蛋白-HIV-1相互作用，这将为开发针对HIV-1生命周期中重要步骤的新型抗病毒药物确定目标。性能现场=

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================