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: 10646359
• 负责人: Mamuka Kvaratskhelia
• 金额: $ 56.64万
• 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/10646359
• 关键词: Amino Acids; Antiviral Agents; Binding; Biochemical; Biochemistry; Biological Assay; Biophysics; Capsid; Capsid Proteins; Cell Nucleus; Cell fusion; Cells; Complex; Cytoplasm; DNA; DNA Integration; Data; Dyes; Electron Microscopy; Event; Exclusion; 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; Productivity; 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 运输到核斑点，然后病毒 DNA 整合到宿主基因组中。在之前的某个时刻 整合时，病毒核糖核蛋白复合物周围的衣壳壳必须分解（脱壳）以 释放病毒预整合复合物。目前尚不清楚细胞脱壳发生在何处以及是否发生 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反应和功能性病毒复合物的核输入至关重要。 几个小组根据 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间接方法的使用 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 脱壳是一个多步骤过程，涉及衣壳壳的透化 细胞质、核孔重塑以及细胞核中 CA 的丢失。我们将使用一种新颖的微创技术 直接 CA 标记策略，基于非规范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 核心透化和脱壳事件 感染（目标 1）。另一个知识空白涉及导致 HIV-1 整合的脱涂层后过程 以及发生整合的亚核区室。我们假设病毒预整合复合物 从衣壳壳分离并行进到核斑点的边缘，在那里它与整合酶接合 结合宿主因子 LEDGF/p75 以整合到宿主基因组中。我们将采用一种新型活细胞单病毒 DNA 可视化技术可追踪单个病毒复合物的核运输和生产性整合 建立主动转录病毒 RNA 焦点（目标 2）。最后，我们将使用强大的生化面板， 生物物理学、结构生物学、病毒学和显微镜技术来表征新型 HIV-1 CA 结合 宿主因子 RBM14，我们假设它可以调节核输入后感染的预整合步骤 （目标 3）。这些实验有望阐明有争议的 HIV-1 脱壳过程，揭示 导致生产性整合和整合地点的动态事件，以及描述 RBM14-的作用 早期感染中衣壳相互作用，从而为新的抗病毒策略提供信息。

项目成果

A Novel Phenotype Links HIV-1 Capsid Stability to cGAS-Mediated DNA Sensing.

一种新表型将 HIV-1 衣壳稳定性与 cGAS 介导的 DNA 传感联系起来。

• DOI: 10.1128/jvi.00706-19
• 发表时间: 2019
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Siddiqui,MohammadAdnan;Saito,Akatsuki;Halambage,UpulD;Ferhadian,Damien;Fischer,DouglasK;Francis,AshwanthC;Melikyan,GregoryB;Ambrose,Zandrea;Aiken,Christopher;Yamashita,Masahiro
• 通讯作者: Yamashita,Masahiro

HIV-1 Capsid Uncoating Is a Multistep Process That Proceeds through Defect Formation Followed by Disassembly of the Capsid Lattice.

• DOI: 10.1021/acsnano.3c07678
• 发表时间: 2024-01-30
• 期刊: ACS NANO
• 影响因子: 17.1
• 作者: Gifford, Levi B.;Melikyan, Gregory B.
• 通讯作者: Melikyan, Gregory B.

Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells.

• DOI: 10.1371/journal.ppat.1005709
• 发表时间: 2016-06
• 期刊: PLoS pathogens
• 影响因子: 6.7
• 作者: Francis AC;Marin M;Shi J;Aiken C;Melikyan GB
• 通讯作者: Melikyan GB

HIV-1 Uncoating and Nuclear Import Precede the Completion of Reverse Transcription in Cell Lines and in Primary Macrophages.

• DOI: 10.3390/v12111234
• 发表时间: 2020-10-30
• 期刊: Viruses
• 作者: Francis AC;Marin M;Prellberg MJ;Palermino-Rowland K;Melikyan GB
• 通讯作者: Melikyan GB

Multidisciplinary studies with mutated HIV-1 capsid proteins reveal structural mechanisms of lattice stabilization.

• DOI: 10.1038/s41467-023-41197-7
• 发表时间: 2023-09-12
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Gres AT;Kirby KA;McFadden WM;Du H;Liu D;Xu C;Bryer AJ;Perilla JR;Shi J;Aiken C;Fu X;Zhang P;Francis AC;Melikyan GB;Sarafianos SG
• 通讯作者: Sarafianos SG