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 美国大约有 120 万人，全世界大约有 4000 万人感染艾滋病毒。在 尽管艾滋病毒/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 感染的选项。 ART 在控制病毒和制造 HIV 方面非常有效 感染是一种可控制的疾病。具体而言，HIV-1整合酶（IN）抑制剂为首选药物 在当前的 ART 治疗方案中。然而，这些抑制剂继续面临耐药性，并且越来越多 人们担心这些抑制剂的不良代谢和神经精神作用。因此，需要 开发新的和改进的IN抑制剂以有效和长期控制HIV-1感染。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 对从急性感染细胞中提取的预整合复合物（PIC）的研究有助于 定义 HIV-1 整合机制。此外，PIC研究在 目前使用的 IN 抑制剂的开发。因此，我们建议定义 HIV-1 PIC 的机制 形成将产生新知识，以促进新的和改进的基于 IN 的疗法的开发。 逆转录病毒复制依赖于逆转录（RTN）和整合的基本步骤。 就 HIV 而言，RNA 基因组通过逆转录被逆转录为 DNA 副本 复杂（RTC）。然后，RTC 通过未知机制和未定义时间转换为 PIC。图片， 包含病毒 DNA、IN 酶和其他病毒/宿主因子，进行整合。盛行的 观点认为 HIV-1 RTN 在细胞质和/或核孔复合体 (NPC) 中完成。因此，核 PIC 的进入对于将 HIV-1 DNA 整合到宿主染色体中是必要的。然而，新 有证据表明完整的 HIV-1 衣壳进入细胞核，RTN 在进入细胞核后完成 步。 RTN 的核完成在我们目前对 PIC 的理解中造成了关键的知识差距。我们 假设功能性 HIV-1 PIC 在 RTN 完成之前形成，并且核进入保护 PIC 病毒 DNA 的完整性。该提案将通过三个具体目标来检验这一假设： 目标 1 将 定义功能性 PIC 形成时间与 RTN 完成之间的联系，目标 2 将评估 核进入对 PIC 功能的作用，目标 3 将决定 RTC 到 图片。为了这些目标，我们开发了一种量化 PIC 特定集成活动的新方法， 测量 HIV-1 核心介导的 DNA 整合，以及病毒进入核的阻断和时间 抑制剂添加测定。这些研究将共同​​确定 HIV-1 之间的机制和动力学联系 RTN和PIC功能。最重要的是，我们的团队具有独特的资格，能够及时成功地完成这一任务 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（Campbell）和整合（Engelman 和 Craigie）。