A Proteomic and Biochemical Study of HIV-1 Nucleoprotein Complexes

HIV-1 核蛋白复合物的蛋白质组学和生化研究

• 批准号: 8068257
• 负责人: MICHAEL A BELSHAN
• 金额: $ 35.86万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8068257
• 关键词: Affinity; Affinity Chromatography; Anti-Retroviral Agents; Antiviral Therapy; Area; Biochemical; Biological; Biological Assay; Biotinylation; Cell Nucleus; Cell physiology; Cells; Centrifugation; Combination Drug Therapy; Complement; Complex; Cyclophilin A; DNA; Detection; Development; Drug Delivery Systems; Drug resistance; Effectiveness; Event; Funding; G22P1 gene; Goals; HIV; HIV-1; Highly Active Antiretroviral Therapy; Individual; Infection; Investigation; Knowledge; Mass Spectrum Analysis; Measures; Methods; Morbidity - disease rate; Multi-Drug Resistance; Nuclear Import; Nucleoproteins; Play; Preparation; Prevalence; Probability; Protein Family; Proteins; Proteomics; Qualitative Methods; RNA Binding; Reporting; Repression; Research; Resistance; Reverse Transcription; Role; Sampling; System; Therapeutic; Toxic effect; Transcriptional Regulation; Viral; Viral Genome; Virion; Virus; Virus Replication; drug discovery; effective therapy; gag-pol Fusion Proteins; gain of function; in vivo; inhibitor/antagonist; innovation; knock-down; loss of function; member; mortality; novel; protein complex; public health relevance; resistant strain; small hairpin RNA; therapeutic target; therapy development; viral DNA; viral RNA

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.

描述（由申请人提供）：人类免疫缺陷病毒1型（HIV-1）的耐药菌菌株对有效的长期感染构成了重大挑战。一种对病毒抗性菌株的相对措施是针对新型HIV-1复制领域的治疗方法。我们研究的长期目标是阐明HIV-1前整合络合物（PIC）的组装和运输，以帮助开发新型的抗逆转录病毒疗法。 PIC通过将病毒DNA靶向逆转录后的细胞核，促进了细胞对HIV-1的感染。由于在产生和净化足够数量的图片方面面临挑战，因此对它们的组装，组成和核进口机理的理解有限。因此，既没有现有的图片抑制剂，也没有任何开发中的抑制剂。该建议的目的是识别和表征HIV-1图片的新细胞成分。我们已经开发了两种互补方法，以可重复生成足够数量的功能图片，以通过质谱分析。通过成功检测已知的HIV-1 PIC成分和几种候选蛋白的鉴定，这些方法的可行性证明了这些方法的可行性。到目前为止，至少有一名候选者是LRP130，对HIV-1感染至关重要。这些提出的研究将继续研究LRP130和其他候选蛋白在HIV-1复制中的作用，使用功能障碍和功能丧失测定法。我们还将使用两种创新策略确定其他与PIC相关的细胞蛋白。首先，我们将对通过速度梯度离心纯化的图片进行定量的2-DIGE蛋白质组学分析。其次，我们将使用一种新型的体内生物素化方法来纯化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，提高对PIC组装和运输的理解，并发现开发抗病毒疗法的新目标。具体目的是：1。确定已确定的候选蛋白在HIV-1复制中的作用。 2。在早期复制过程中确定与HIV-1图片相关的其他细胞蛋白。 3。表征新的候选蛋白。 公共卫生相关性：HIV-1快速发展对高度活跃抗逆转录病毒疗法组成部分的抗性的能力对成功的长期治疗受感染的个体构成了重大挑战。针对HIV-1复制领域的新药物具有持续抑制病毒复制的最大可能性。该提案的目的是识别和表征与HIV-1前整合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。