Imaging early steps of HIV-1 infection and virus-host factor interactions

HIV-1 感染的早期成像和病毒-宿主因子相互作用

• 批准号: 10548587
• 负责人: Mamuka Kvaratskhelia
• 金额: $ 58.21万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-17 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10548587
• 关键词: Amino Acids; Antiviral Agents; Binding; Biochemical; Biochemistry; Biological Assay; Biophysics; Capsid; Capsid Proteins; Cell Nucleus; Cell fusion; Cells; Complex; Conflict (Psychology); Cytoplasm; DNA; DNA Integration; Data; Dyes; Electron Microscopy; Event; Genes; Genetic Transcription; Genome; HIV; HIV-1; Image; Imaging Device; Imaging Techniques; Imaging technology; In Vitro; Infection; Innate Immune Response; Integrase; Integration Host Factors; Knowledge; Label; Light; Location; Macromolecular Complexes; Maps; Mass Spectrum Analysis; Microscopy; Modeling; Nuclear; Nuclear Import; Nuclear Pore; Permeability; Process; Proteins; Recombinants; Regulation; Reporting; Reverse Transcription; Ribonucleoproteins; Role; Site; Techniques; Technology; Travel; Tube; Validation; Viral; Virus; Visualization; base; experimental study; integration site; knock-down; live cell imaging; minimally invasive; novel; novel strategies; novel virus; nucleocytoplasmic transport; recruit; stem; structural biology; transcriptional coactivator p75; viral DNA; viral RNA; virology

Key steps of early HIV-1 infection include reverse transcription, nuclear import of replication complexes and transport to nuclear speckles followed by viral DNA integration into host genome. At some point before integration, the capsid shell surrounding the viral ribonucleoprotein complex must disassemble (uncoat) to release the viral pre-integration complexes. It is currently unclear where in the cell uncoating occurs and whether the capsid (CA) protein is progressively or synchronously lost from the capsid shell, yet optimal core stability is essential for evading the host innate immune responses and for nuclear import of functional viral complexes. Highly divergent findings regarding HIV-1 uncoating have been reported by several groups based upon the visualization of single virus uncoating in live cells. These conflicting results stem, in part, from the use of indirect CA labeling approaches and lack of minimally invasive direct fluorescent labeling of HIV-1 capsid. We hypothesize that HIV-1 uncoating is a multi-step process that involves permeabilization of the capsid shell in the cytoplasm, remodeling at the nuclear pore, and loss of CA in the nucleus. We will use a novel minimally invasive direct CA labeling strategy, which is based on site-directed incorporation of non-canonical amino acids and click- labeling with an organic dye, to elucidate single HIV-1 core permeabilization and uncoating events resulting to infection (Aim 1). Another gap in knowledge pertains to post-uncoating processes leading to HIV-1 integration and sub-nuclear compartments where integration occurs. We hypothesize that the viral pre-integration complex separates from the capsid shell and travels to the edge of a nuclear speckle where it engages the integrase- binding host factor LEDGF/p75 for integration into host genome. We will employ a novel live-cell single viral DNA visualization technology to track nuclear transport and productive integration of single viral complexes that establish actively transcribing viral RNA foci (Aim 2). Finally, we will use a powerful panel of biochemical, biophysical, structural biology, virology, and microscopy techniques to characterize a novel HIV-1 CA binding host factor, RBM14, which we hypothesize to modulate pre-integration steps of infection after nuclear import (Aim 3). These experiments are expected to elucidate the controversial HIV-1 uncoating process, reveal the dynamic events leading to productive integration and sites of integration, as well as delineate the role of RBM14- capsid interactions in early infection, thus informing novel antiviral strategies.

早期HIV-1感染的关键步骤包括逆转录、复制复合物的核输入和 转运到核斑点，随后病毒DNA整合到宿主基因组中。在之前的某个时候 整合时，病毒核糖核蛋白复合物周围的衣壳必须分解（脱壳）， 释放病毒整合前复合物目前尚不清楚细胞中的何处发生脱膜，以及 衣壳（CA）蛋白逐渐或同步地从衣壳壳中丢失，但最佳的核心稳定性是 对于逃避宿主先天免疫应答和功能性病毒复合物的核输入是必需的。 几个研究小组报告了关于HIV-1脱壳的高度分歧的发现， 活细胞中单个病毒脱壳的可视化。这些相互矛盾的结果部分源于使用间接 CA标记方法和缺乏HIV-1衣壳的微创直接荧光标记。我们 假设HIV-1去包被是一个多步骤的过程，包括在细胞膜中衣壳壳的透化， 细胞质中，在核孔的重塑，和CA在细胞核中的损失。我们将使用一种新的微创 直接CA标记策略，其基于非典型氨基酸的定点掺入和点击， 用有机染料标记，以阐明单个HIV-1核心透化和去包被事件， 感染（目标1）。另一个知识空白涉及到导致HIV-1整合的去包被后过程 以及整合发生的亚核区室。我们假设病毒整合前复合体 从衣壳壳分离，并移动到核斑点的边缘，在那里它与整合酶结合， 结合宿主因子LEDGF/p75以整合到宿主基因组中。我们将采用一种新的活细胞单病毒DNA 可视化技术来跟踪单个病毒复合物的核运输和生产性整合， 建立活跃转录的病毒RNA灶（Aim 2）。最后，我们将使用一个强大的生化小组， 生物物理学、结构生物学、病毒学和显微镜技术来表征一种新的HIV-1 CA结合 宿主因子RBM 14，我们假设其调节核输入后感染的整合前步骤 (Aim 3）。这些实验有望阐明有争议的HIV-1脱壳过程，揭示 导致生产性一体化的动态事件和一体化地点，并说明成果管理制的作用14- 衣壳相互作用在早期感染，从而通知新的抗病毒策略。