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: 8260860
• 负责人: MICHAEL A BELSHAN
• 金额: $ 35.87万
• 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/8260860
• 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）：1 型人类免疫缺陷病毒 (HIV-1) 的耐药株对感染的长期有效治疗提出了重大挑战。针对耐药病毒株的一种对策是针对 HIV-1 复制的新区域进行治疗。我们研究的长期目标是阐明 HIV-1 预整合复合物 (PIC) 的组装和运输，以帮助开发新型抗逆转录病毒疗法。 PIC 通过逆转录后将病毒 DNA 靶向细胞核，促进 HIV-1 细胞的感染。由于生产和纯化足够数量的 PIC 方面存在挑战，人们对其组装、组成和核进口机制的了解有限。因此，既没有现有的 PIC 抑制剂，也没有正在开发的抑制剂。该提案的目的是鉴定和表征 HIV-1 PIC 的新型细胞成分。我们开发了两种互补方法，可重复生产足够数量的功能性 PIC，用于质谱分析。成功检测已知的 HIV-1 PIC 成分并鉴定了几种候选蛋白，证明了这些方法的可行性。迄今为止，至少有一种候选药物 LRP130 已被发现对 HIV-1 感染至关重要。这些拟议的研究将使用功能获得和功能丧失分析继续研究 LRP130 和其他候选蛋白在 HIV-1 复制中的作用。我们还将使用两种创新策略来鉴定其他 PIC 相关细胞蛋白。首先，我们将对速度梯度离心纯化的 PIC 进行定量 2-D-DiGE 蛋白质组分析。其次，我们将使用一种新颖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 复制早期步骤至关重要的新细胞辅助因子，增进对 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. 确定已确定的候选蛋白在 HIV-1 复制中的作用。 2. 鉴定早期复制过程中与 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 能够迅速对高效抗逆转录病毒疗法的成分产生耐药性，这对感染者的成功长期治疗构成了重大挑战。针对 HIV-1 复制新区域的新药最有可能持续抑制病毒复制。该提案的目标是鉴定和表征与 HIV-1 预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。