A Proteomic and Biochemical Study of HIV-1 Nucleoprotein Complexes

描述（由申请方提供）：人类免疫缺陷病毒1型（HIV-1）的耐药菌株对有效的长期感染治疗构成了重大挑战。针对耐药病毒株的一种对策是用靶向HIV-1复制的新区域的疗法进行治疗。我们研究的长期目标是阐明HIV-1前整合复合物（PIC）的组装和转运，以帮助开发新的抗逆转录病毒疗法。PIC通过在逆转录后将病毒DNA靶向到细胞核中来促进HIV-1感染细胞。由于生产和纯化足够量的PIC的挑战，对其组装、组成和核输入机制的理解有限。因此，既没有现有的PIC抑制剂，也没有任何正在开发的抑制剂。该提案的目的是识别和表征HIV-1 PIC的新细胞成分。我们已经开发了两种互补的方法，可重复地产生足够数量的功能PIC用于质谱分析。这些方法的可行性证明了已知的HIV-1 PIC组件的成功检测和几个候选蛋白的鉴定。到目前为止，至少有一种候选基因LRP 130被发现对HIV-1感染至关重要。这些拟议的研究将继续调查LRP 130和其他候选蛋白在HIV-1复制中的作用，使用功能获得和功能丧失试验。我们还将使用两种创新策略鉴定其他PIC相关细胞蛋白。首先，我们将进行定量2-D-DiGE蛋白质组学分析的速度梯度离心纯化的PIC。其次，我们将使用一种新的体内生物素化方法亲和纯化HIV-1蛋白复合物。这些研究的成功完成将确定对HIV-1复制的早期步骤至关重要的新的细胞辅助因子，推进对PIC组装和运输的理解，并发现开发抗病毒疗法的新靶点。具体目标是：1。确定已鉴定的候选蛋白在HIV-1复制中的作用。 2.确定在早期复制过程中与HIV-1 PIC相关的其他细胞蛋白。 3.表征新的候选蛋白质。 公共卫生相关性：HIV-1能够迅速发展出对高效抗逆转录病毒疗法组成部分的耐药性，这对受感染者的成功长期治疗构成了重大挑战。靶向HIV-1复制新区域的新药最有可能持续抑制病毒复制。该提案的目标是识别和表征与HIV-1整合前复合物相关的候选细胞蛋白，以开发新的抗逆转录病毒疗法。

DESCRIPTION (provided by applicant): Drug resistant strains of human immunodeficiency virus type 1 (HIV-1) pose a significant challenge for effective long-term treatment of infection. One counter-measure against resistant strains of virus is treatment with therapeutics targeted to novel areas of HIV-1 replication. The long term goal of our research is to elucidate the assembly and transport of the HIV-1 preintegration complex (PIC) to aid in the development of novel antiretroviral therapeutics. The PIC facilitates the infection of cells with HIV-1 by targeting the viral DNA into the nuclei of cells after reverse transcription. Due to challenges in producing and purifying sufficient quantities of PICs there is limited understanding of their assembly, composition, and mechanism of nuclear import. Consequently, there are neither existing inhibitors of PICs nor any in development. The objective of this proposal is to identify and characterize novel cellular components of HIV-1 PICs. We have developed two complementary methods to reproducibly produce sufficient quantities of functional PICs for analysis by mass spectrometry. The feasibility of these approaches is demonstrated by the successful detection of known HIV-1 PIC components and the identification of several candidate proteins. At least one candidate thus far, LRP130, has been found to be critical for HIV-1 infection. These proposed studies will continue the investigation of role of LRP130 and other candidate proteins in HIV-1 replication using gain-of-function and loss-of-function assays. We will also identify additional PIC-associated cellular proteins using two innovative strategies. First, we will perform quantitative 2-D-DiGE proteomic analysis on PICs purified by velocity gradient centrifugation. Second, we will affinity purify HIV-1 protein complexes using a novel in vivo biotinylation method. Successful completion of these studies will identify new cellular co-factors critical for the early steps of HIV-1 replication, advance the understanding of PIC assembly and transport, and discover new targets for the development of antiviral therapies. The Specific Aims are: 1. To determine the role of identified candidate proteins in HIV-1 replication. 2. To identify additional cellular proteins associated with HIV-1 PICs during early replication. 3. To characterize new candidate proteins. PUBLIC HEALTH RELEVANCE: The ability of HIV-1 to rapidly evolve resistance to the components of highly active antiretroviral therapy poses a significant challenge for the successful long-term treatment of infected individuals. New drugs that target novel areas of HIV-1 replication have the greatest probability for continued repression of virus replication. The goal of this proposal is to identify and characterize candidate cellular proteins associated with HIV-1 preintegration complexes for the development of new antiretroviral therapeutics.