Dynamics of HIV Nuclear Interactions

HIV核相互作用的动力学

• 批准号: 10650885
• 负责人: Alan N. Engelman
• 金额: $ 42.94万
• 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/10650885
• 关键词: Address; Affect; Bar Codes; Behavior; Binding; Biochemistry; Bioinformatics; 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; Environment; Free Energy; G22P1 gene; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Goals; Growth Factor; 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; Reverse Transcription; Role; Site; Structure; Surface; System; T-Lymphocyte; Technology; Trans-Activators; Transcription Initiation; Viral; Virion; Virus; Virus Integration; antiretroviral therapy; base; biophysical techniques; genome-wide; histone modification; in vivo; insight; integration site; latent HIV reservoir; latent infection; lens epithelium-derived growth factor; migration; molecular dynamics; molecular modeling; mutant; novel; particle; preference; prevent; repaired; response; spatial integration; tat Protein; trafficking; translation assay; 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 逆转录为病毒 DNA (vDNA)，并整合到宿主细胞中 基因组。 vDNA必须进入细胞核才能接触宿主DNA进行整合，但是vDNA如何获得 核进入、迁移到整合位点以及选择整合位点是必不可少的，但人们对此知之甚少 HIV复制的步骤。彻底了解病毒如何利用宿主因素来完成这些关键 需要复制步骤来确定有希望的分子靶标，以开发新的抗逆转录病毒药物 疗法。核输入后，完整的病毒核心被转运到称为核的染色质间结构域。 在它们脱去外壳的地方形成斑点，以便预整合复合物 (PIC) 可以将 vDNA 整合到附近 染色质。在目标 1 中，我们将识别并阐明影响核的动态 HIV 核心-宿主细胞相互作用。 病毒核心的脱壳效率/动力学和核穿透距离；我们还将确定是否 HIV核心-宿主相互作用利用多种协同作用影响全基因组整合位点选择 实验方法。 HIV整合的主要底物是染色质和核小体DNA，并且 整合酶 (IN) 与晶状体上皮的结合有利于整合到转录活性基因中 衍生生长因子（LEDGF）。 HIV PIC 如何与 LEDGF 和核小体相互作用以及哪些靶位点 尚未确定是否有利于纯化组装的 IN-vDNA 复合物（整合体）的整合。在 目标 2，我们将构建体外整合的生理相关染色质靶标，定义首选区域 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/宿主相互作用的机制基础 病毒 DNA 直接整合到染色质中。人们普遍认为整合位点的染色质状态 决定转录活性或进展到潜伏期。然而，宿主染色质状态和病毒因素 控制活动或潜伏感染的建立仍然是个谜。 HIV 潜伏病毒库重新引发感染 在停止抗逆转录病毒治疗后，迄今为止，它们已经阻止了治疗艾滋病毒感染的方法的开发。我们 将开发多种实验方法来阐明建立潜伏感染和 重新启动转录，这可能会促进潜伏期重新激活或潜伏期促进治疗的发展 并实现艾滋病毒感染的根除或功能性治愈。在目标3中，我们将开发多个实验 确定 IN、Tat 和体内整合位点作用的系统，以检查整合位点的作用 染色质在建立主动感染与潜伏感染中的作用。总的来说，这些研究将提供变革性的见解 HIV脱壳、整合位点选择、指导整合到核小体的分子机制 DNA，并阐明整合位点选择对 HIV 转录能力的功能影响 和延迟。