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120万人感染艾滋病毒，全球约有4000万人感染。在……里面 尽管艾滋病毒/艾滋病研究取得重大进展，但抗逆转录病毒疗法(ART)仍然是唯一的治疗方法。 可用于HIV-1感染的选项。抗逆转录病毒疗法在控制病毒和制造艾滋病毒方面非常有效。 感染是一种可以控制的疾病.具体地说，HIV-1整合酶抑制剂(IN)被用作首选药物 在当前的艺术养生法中。然而，这些抑制剂继续面临抗药性，而且有越来越多的 关注这些抑制剂的不良代谢和神经精神影响。因此，有必要对 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。这样的努力 关键取决于对艾滋病毒-1整合机制的更好理解。值得注意的是， 对从急性感染细胞中提取的整合前复合体(PIC)的研究在 明确HIV-1整合的机制。此外，太平洋岛屿国家的研究在 目前使用的IN抑制剂的研究进展。因此，我们提出的定义HIV-1 PIC机制的建议 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)和整合这两个关键步骤。 在艾滋病毒的情况下，rna基因组通过逆转录反转录成dna副本。 复合体(RTC)。然后，RTC以未知的机制和未知的时间转变为PIC。PIC， 包含病毒DNA、IN酶和其他病毒/宿主因子，进行整合。盛行的 有观点认为，HIV-1RTN是在细胞质和/或核孔复合体(NPC)中完成的。因此，核 PIC的进入是HIV-1DNA整合到宿主染色体中所必需的。然而，新的 证据表明，完整的HIV-1衣壳进入细胞核，RTN在核进入后完成 一步。RTN的核建成在我们目前对PIC的理解中造成了关键的知识空白。我们 假定HIV-1功能PIC在RTN完成之前形成，并且核进入保护 PIC病毒DNA的完整性。这项提议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向 图为。为了这些目标，我们开发了一种新的方法来量化特定于PIC的整合活动和 测量HIV-1核心介导的DNA整合，以及阻断病毒核进入和时间 抑制物添加试验。总而言之，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及时成功完成这项工作的人 R01-项目，由于我们拥有研究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)。