Spatiotemporal Staging of the HIV-1 Preintegration complex

HIV-1 预整合复合体的时空分期

• 批准号: 10548553
• 负责人: Chandravanu Dash
• 金额: $ 65.88万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-20 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10548553
• 关键词: AIDS/HIV problem; Acute; Affect; Anti-Retroviral Agents; Biochemical; Biochemistry; Biological Assay; Biology; Capsid; Cell Nucleus; Cells; Chromosomes; Complex; Coupled; Crude Extracts; Cytoplasm; DNA; DNA Integration; DNA biosynthesis; Data; Development; Disease Management; Drug resistance; Enzymes; Face; Genome; HIV; HIV Infections; HIV-1; HIV-1 integrase; Hour; In Vitro; Infection; Integrase; Integrase Inhibitors; Integration Host Factors; Kinetics; Knowledge; Life; Link; Measures; Mediating; Metabolic; Modeling; Monitor; Nuclear; Nuclear Extract; Nuclear Pore Complex; Persons; Pharmaceutical Preparations; Play; Positioning Attribute; RNA; Reagent; Regimen; Research; Reverse Transcription; Role; Staging; T-Lymphocyte; Testing; Time; Transcription Process; Viral; Virus; Virus Integration; antiretroviral therapy; base; drug development; ds-DNA; improved; in vitro activity; inhibitor; insertion/deletion mutation; mutant; neuropsychiatry; novel; novel strategies; preservation; spatiotemporal; time use; tool; viral DNA; viral RNA

ABSTRACT Approximately 1.2 million people in the US and ~40 million people worldwide are infected with HIV. In spite of significant progress in HIV/AIDS research, anti-retroviral therapy (ART) remains the only treatment option available for HIV-1 infection. ART has been highly effective in controlling the virus and making HIV infection a manageable disease. Specifically, inhibitors of HIV-1 Integrase (IN) are used as the preferred drugs in the current ART regimens. However, these inhibitors continue to face drug resistance and there is growing concern of the adverse metabolic and neuropsychiatric effects of these inhibitors. Thus, there is a need for the development of new and improved IN inhibitors for effective and long-term control HIV-1 infection. Such effort is critically dependent on a better understanding of the mechanisms of HIV-1 integration. It is important to note that studies of the preintegration complex (PIC) extracted from acutely infected cells have been instrumental in defining the mechanisms of HIV-1 integration. Furthermore, PIC studies have played a central role in the development of currently used IN inhibitors. Therefore, our proposal to define the mechanism of HIV-1 PIC formation will generate new knowledge to promote the development of new and improved IN-based therapies. Retroviral replication is dependent on the essential steps of reverse transcription (RTN) and integration. In the case of HIV, the RNA genome is reverse transcribed into a DNA copy by the reverse transcription complex (RTC). Then, the RTC transitions into a PIC by an unknown mechanism and undefined time. The PIC, containing the viral DNA, the IN enzyme, and other viral/host factors, carries out integration. The prevailing view is that HIV-1 RTN is completed in the cytoplasm and/or at the nuclear pore complex (NPC). Thus, nuclear entry of the PIC is necessary to carry out HIV-1 DNA integration into the host chromosomes. However, new evidence suggest that the intact HIV-1 capsid enters the nucleus and RTN is completed after the nuclear entry step. A nuclear completion of RTN creates key knowledge gaps in our current understanding of the PIC. We hypothesize that functional HIV-1 PICs are formed prior to the completion of RTN and nuclear entry protects the integrity of the PIC-viral DNA. This proposal will test this hypothesis through three specific aims: Aim 1 will define the link between the timing of functional PIC formation relative to RTN completion, Aim 2 will assess the role of nuclear entry on PIC function, and Aim 3 will determine the spatiotemporal transition of the RTC to a PIC. For these aims, we have developed a novel approach of quantifying PIC-specific integration activity and measuring HIV-1 core-mediated DNA integration, coupled with the blockade of viral nuclear entry and time of inhibitor addition assays. Together, these studies will define the mechanistic and kinetic link between HIV-1 RTN and PIC function. Most importantly, our team is uniquely qualified to successfully complete this timely R01-project, since we have the expertise, reagents, and the tools required to study PIC biochemistry (Dash), Capsid biology (Aiken), Nuclear entry (Campbell), and Integration (Engelman and Craigie).

抽象的 在美国，大约有120万人和全世界约4000万人感染了艾滋病毒。在 艾滋病毒/艾滋病研究取得了重大进展，抗逆转录病毒疗法（ART）仍然是唯一的治疗方法 可用于HIV-1感染的选件。艺术在控制病毒和艾滋病毒方面非常有效 感染可控制的疾病。具体而言，HIV-1整合酶的抑制剂（in）用作首选药物 在当前的艺术方案中。但是，这些抑制剂继续面临耐药性，并且正在增长 关注这些抑制剂的不良代谢和神经精神作用。因此，需要 在有效和长期对照HIV-1感染中开发新的和改善的抑制剂。这样的努力 关键取决于对HIV-1整合机制的更好理解。重要的是要注意 从急性感染细胞中提取的预一体化复合物（PIC）的研究在 定义HIV-1整合的机制。此外，图片研究在 当前用于抑制剂的开发。因此，我们定义HIV-1 PIC机制的建议 形成将产生新知识，以促进基于新的疗法的新知识和改进。 逆转录病毒复制取决于逆转录（RTN）和集成的基本步骤。 在HIV的情况下，RNA基因组通过逆转录将RNA基因组反转录为DNA拷贝 复杂（RTC）。然后，RTC通过未知的机制和未定义的时间过渡到PIC。图片， 包含病毒DNA，IN酶和其他病毒/宿主因子进行整合。盛行 观点是HIV-1 RTN在细胞质和/或核孔复合物（NPC）中完成。因此，核 对于将HIV-1 DNA整合到宿主染色体中是必要的。但是，新的 有证据表明，完整的HIV-1衣壳进入核和RTN，在核进入后完成 步。 RTN的核能完成在我们当前对图片的理解中造成了关键的知识差距。我们 假设功能性HIV-1图片是在RTN完成之前形成的，核进入保护 PIC-病毒DNA的完整性。该建议将通过三个特定目的来检验这一假设：目标1将 定义相对于RTN完成的功能PIC形成时间之间的联系，AIM 2将评估 核进入对PIC功能的作用，AIM 3将决定RTC的时空过渡 图片。对于这些目的，我们开发了一种量化特定于PIC特定整合活动和的新方法 测量HIV-1核心介导的DNA整合，再加上病毒核进入的阻塞和 抑制剂添加测定。总之，这些研究将定义HIV-1之间的机械和动力学联系 RTN和PIC功能。最重要的是，我们的团队具有独特的资格，可以成功完成此及时完成 R01项目，因为我们拥有研究PIC生物化学（DASH）所需的专业知识，试剂和工具 衣壳生物学（Aiken），核进入（坎贝尔）和整合（恩格曼和克雷吉）。