Dynamics of HIV Nuclear Interactions

HIV核相互作用的动力学

• 批准号: 10508451
• 负责人: Alan N. Engelman
• 金额: $ 38.2万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-22 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10508451
• 关键词: Address; Affect; Bar Codes; Behavior; Binding; Biochemistry; Bioinformatics; Biological Assay; Biophysics; C-terminal; CD4 Positive T Lymphocytes; Cell Communication; Cell Line; Cell Nucleus; Cells; Cellular biology; Chemistry; Chimeric Proteins; Chromatin; Clinical; Clinical Virology; Competence; Complex; Computer Models; Cryoelectron Microscopy; DNA; DNA Integration; DNA Repair Enzymes; Development; Electron Microscopy; Environment; Free Energy; G22P1 gene; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Goals; HIV; HIV Infections; HIV-1; Human Genome; Image; Imaging Techniques; In Vitro; Individual; Infection; Integrase; Integration Host Factors; Kinetics; Knowledge; Laboratories; Laboratory Research; Link; Location; Molecular; Molecular Probes; Molecular Target; Nuclear; Nuclear Accidents; Nuclear Import; Nucleoplasm; Nucleosomes; Penetration; Physiological; Positioning Attribute; Process; Production; Protein Chemistry; Proteomics; Proviruses; RNA; Reporter; Research; Resolution; Role; Site; Structure; Surface; System; T-Lymphocyte; Technology; Trans-Activators; Viral; Virion; Virus; Virus Integration; antiretroviral therapy; base; biophysical techniques; cryogenics; genome-wide; histone modification; in vivo; insight; integration site; latent HIV reservoir; latent infection; lens epithelium-derived growth factor; molecular dynamics; molecular modeling; mutant; novel; particle; preference; prevent; repaired; response; spatial integration; tat Protein; trafficking; viral DNA; virology

ABSTRACT, PROJECT 2 After infecting a cell, HIV reverse transcribes its RNA into viral DNA (vDNA) that integrates into the host cell genome. The vDNA must enter the nucleus to access the host DNA for integration, but how the vDNA gains nuclear entry, migrates to sites of integration, and selects sites of integration are essential yet poorly understood steps in HIV replication. A thorough understanding of how the virus exploits host factors to complete these key steps in replication is needed to identify promising molecular targets for development of new antiretroviral therapies. Following nuclear import, intact viral cores are transported to interchromatin domains called nuclear speckles where they uncoat so that the preintegration complex (PIC) can integrate the vDNA into nearby chromatin. In Aim 1, we will identify and elucidate dynamic HIV core-host cell interactions that influence nuclear uncoating efficiency/kinetics and the nuclear penetration distance of viral cores; we will also determine whether the HIV core-host interactions affect genome-wide selection of integration sites using multiple synergistic experimental approaches. The main substrate for HIV integration is chromatin and nucleosomal DNA, and integration into transcriptionally active genes is favored by association of integrase (IN) with lens epithelium derived growth factor (LEDGF). How HIV PICs interact with LEDGF and nucleosomes and which target sites are favored for integration by purified assembled IN-vDNA complexes (intasomes) has not yet been determined. In Aim 2, we will construct physiologically relevant chromatin targets of integration in vitro, define preferred regions for viral integration into nucleosome substrates and elucidate how host factors and chromatin states modulate integration site preferences. These directions will elucidate the mechanistic bases by which viral/host interactions direct viral DNA integration into chromatin. It is generally thought that the chromatin state at integration sites determines transcriptional activity or progression to latency. However, host chromatin states and viral factors that control establishment of active or latent infection remain enigmatic. As HIV latent reservoirs reinitiate infection upon cessation of antiretroviral therapy, they have thus far prevented development of a cure for HIV infection. We will develop multiple experimental approaches to elucidate mechanisms that establish latent infection and reinitiate transcription, which may promote development of latency-reactivating or latency promoting treatments and achieve eradication or a functional cure for HIV infection. In Aim 3, We will develop multiple experimental systems to determine the roles of IN, Tat and in vivo integration sites to examine the role of the integration site chromatin in establishment of active vs. latent infection. Overall, these studies will provide transformative insights into HIV uncoating, integration site selection, the molecular machinery that directs integration into nucleosomal DNA, and elucidate the functional consequences of integration site selection on HIV transcriptional competency and latency.

摘要，项目2 感染细胞后，HIV将其RNA反转录为病毒DNA（VDNA），该病毒DNA（VDNA）整合到宿主细胞中 基因组。 VDNA必须进入核以访问宿主DNA以进行整合，但VDNA如何增长 核进入，迁移到整合地点以及整合的选择位置是必不可少的尚不理解的 艾滋病毒复制的步骤。对病毒如何利用宿主因素完成这些密钥的透彻理解 需要复制步骤来识别有希望的分子靶标，以开发新的抗逆转录病毒 疗法。核进口后，完整的病毒核心被运输到称为核的染色质域 斑点它们不适中的位置，以便预一体化复合物（PIC）可以将VDNA整合到附近 染色质。在AIM 1中，我们将确定并阐明影响核的动态HIV核心宿主相互作用 解除效率/动力学和病毒核的核穿透距离；我们还将确定是否 HIV核心宿主相互作用会使用多个协同作用，影响整合位点的全基因组选择 实验方法。 HIV整合的主要底物是染色质和核小体DNA，并且 集成酶（in）与透镜上皮的缔合中的集成在转录活性基因中 得出的生长因子（LEDGF）。 HIV图片如何与LEDGF和核小体相互作用以及哪些目标位点是 尚未确定通过纯化的组装vDNA复合物（IntoSomes）积分的积分。在 AIM 2，我们将在体外构建与生理相关的染色质靶标，定义优先区域 用于病毒整合到核小体底物中，并阐明宿主因子和染色质状态如何调节 集成站点偏好。这些方向将阐明病毒/宿主相互作用的机械基础 直接病毒DNA整合到染色质中。通常认为集成位点的染色质状态 确定转录活性或延迟的进展。但是，宿主染色质状态和病毒因素 主动感染或潜在感染的控制建立仍然神秘。作为艾滋病毒潜在储层重新发明感染 停止抗逆转录病毒疗法后，迄今为止，它们已经阻止了治愈HIV感染的治疗方法。我们 将开发多种实验方法来阐明建立潜在感染和的机制 重新启动转录，这可能促进延迟反应或延迟促进治疗的发展 并获得根除或用于HIV感染的功能治疗。在AIM 3中，我们将开发多个实验 确定IN，TAT和体内整合位点的作用的系统以检查集成位点的作用 建立活性与潜在感染的染色质。总体而言，这些研究将提供变革性的见解 进入艾滋病毒脱落，整合位点的选择，这是指导整合核小体的分子机制 DNA，并阐明集成位点选择对HIV转录能力的功能后果 和延迟。