Spatiotemporal Staging of the HIV-1 Preintegration complex

HIV-1 预整合复合体的时空分期

• 批准号: 10625528
• 负责人: Chandravanu Dash
• 金额: $ 80.56万
• 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/10625528
• 关键词: Acute; Affect; Anti-Retroviral Agents; Biochemical; Biochemistry; Biological Assay; Biology; Capsid; Cell Nucleus; Cells; Chromosomes; Complex; Coupled; Creativeness; Cytoplasm; DNA; DNA Integration; DNA biosynthesis; Data; Development; Disease; Drug resistance; Enzymes; Genome; HIV; HIV Infections; HIV-1; HIV-1 integrase; HIV/AIDS; Hour; In Vitro; Infection; Integrase; Integrase Inhibitors; Integration Host Factors; Kinetics; Knowledge; Link; Measures; Mediating; Metabolic; Modeling; Monitor; Nuclear; Nuclear Extract; Nuclear Pore Complex; Persons; Pharmaceutical Preparations; Play; Positioning Attribute; Qualifying; RNA; Reagent; Regimen; Research; Reverse Transcription; Role; Staging; T-Lymphocyte; Testing; Time; Transcription Process; Viral; Virus; Virus Integration; antiretroviral therapy; drug development; ds-DNA; improved; in vitro activity; inhibitor; 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).

摘要 美国约有120万人，全世界约有4000万人感染艾滋病毒。在 尽管艾滋病研究取得了重大进展，但抗逆转录病毒疗法仍然是唯一的治疗方法 HIV-1感染者的选择。抗逆转录病毒疗法在控制病毒和使艾滋病毒 感染是一种可以控制的疾病。具体地，HIV-1整合酶（IN）的抑制剂被用作优选药物 在目前的ART治疗方案中。然而，这些抑制剂继续面临耐药性， 这些抑制剂的不良代谢和神经精神作用的关注。因此，需要 开发新的和改进的IN抑制剂，用于有效和长期控制HIV-1感染。这种努力 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整合前复合物（PIC）的研究有助于 定义HIV-1整合的机制。此外，事先知情同意研究在 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， 形成将产生新的知识，以促进新的和改进的基于IN的疗法的发展。 逆转录病毒复制依赖于逆转录（RTN）和整合的基本步骤。 在HIV的情况下，RNA基因组通过逆转录被逆转录成DNA拷贝， 复合物（RTC）。然后，RTC通过未知的机制和未定义的时间过渡到PIC。PIC， 含有病毒DNA、IN酶和其他病毒/宿主因子的细胞进行整合。现行 HIV-1 RTN在细胞质和/或核孔复合体（NPC）中完成。因此，核 PIC的进入是进行HIV-1 DNA整合到宿主染色体中所必需的。但新 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空白。我们 假设功能性HIV-1 PIC在RTN完成之前形成，核进入保护了 PIC病毒DNA的完整性。本提案将通过三个具体目标来检验这一假设： 定义功能PIC形成时间与RTN完成时间之间的联系，目标2将评估 核进入对PIC功能的作用，目标3将决定RTC到PIC功能的时空转变 PIC.为了实现这些目标，我们开发了一种新的方法来量化PIC特异性整合活性， 测量HIV-1核心介导的DNA整合，加上病毒核进入的阻断和 抑制剂添加测定。总之，这些研究将确定HIV-1之间的机制和动力学联系， RTN和PIC功能。最重要的是，我们的团队是唯一有资格成功完成这一及时 R 01-项目，因为我们拥有研究PIC生物化学所需的专业知识，试剂和工具（Dash）， 衣壳生物学（Aiken）、核进入（坎贝尔）和整合（Engelman和Craigie）。