Molecular Mechanisms of Retroviral Gag-RNA interactions in Virus Assembly

病毒组装中逆转录病毒 Gag-RNA 相互作用的分子机制

• 批准号: 10656231
• 负责人: Leslie J Parent
• 金额: $ 42.81万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-20 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656231
• 关键词: Address; Adopted; Animals; Automobile Driving; Binding; Biochemical; Biological; Biological Models; Biophysical Process; Biophysics; CRISPR/Cas technology; Cancer Etiology; Cell Nucleus; Cell membrane; Cells; Cellular biology; Chromatin; Complex; Cytoplasm; Cytoplasmic Granules; DNA; Data; Disease; Domestic Fowls; Genetic; Genetic Transcription; Genome; Goals; HIV; Host Defense; Human; Immunologic Deficiency Syndromes; Infection; Integration Host Factors; Intracellular Transport; Kinetics; Knowledge; Laboratories; Liquid substance; Measures; Mediating; Membrane; Modeling; Molecular; Morphogenesis; Nuclear; Nuclear Envelope; Nuclear Export; Organelles; Pathway interactions; Pharmaceutical Preparations; Phase; Physical condensation; Process; Production; Property; Proteomics; Publishing; RNA; RNA Binding; RNA Splicing; RNA Viruses; Research Project Grants; Retroviridae; Retrovirus Proteins; Ribonucleoproteins; Role; Rous sarcoma virus; Site; Source; Structural Protein; Structure; Testing; Thinking; Transcript; Travel; Viral; Virion; Virus Assembly; Work; biophysical analysis; biophysical properties; biophysical techniques; deep sequencing; experimental study; fluorescence imaging; gag Gene Products; genomic RNA; imaging approach; imaging modality; imaging study; innovation; insight; integration site; live cell imaging; microscopic imaging; particle; recruit; single molecule; trafficking; transcription factor; ultra high resolution; viral RNA; viral genomics; virus host interaction

Abstract Retroviruses are positive-sense, single-stranded RNA viruses that cause cancers and severe immunodeficiency diseases in animals and humans, including human immunodeficiency virus. For over a century, Rous sarcoma virus (RSV), which causes cancer in domestic fowl, has served as a powerful model system to dissect the molecular basis of retroviral replication, including retrovirus assembly. Gag, the major structural protein of retroviruses, orchestrates the assembly of virus particles that bud from the plasma membrane of infected cells. To initiate particle assembly, Gag selectively binds unspliced viral RNA as the source of genomic RNA in virions. This proposal focuses on the mechanism by which Gag selects genomic RNA, addressing fundamental, unanswered questions in the field: (i) where in the cell does the initial contact between Gag and viral RNA occur; (ii) how does Gag selectively recruit unspliced viral RNA for packaging when it comprises only ~1% of the total RNA in an infected cell; and (iii) what are the properties of Gag-viral RNA complexes that promote transport through the cell to the plasma membrane for particle release? Because virus particles bud from the plasma membrane, it was originally thought that initial Gag-genomic RNA interactions occurred in the cytoplasm. Our laboratory discovered that RSV Gag undergoes nuclear trafficking, which is required for efficient genomic viral RNA packaging. This finding raised the possibility that Gag binds genomic RNA in the nucleus, which challenges the dogma for how retroviruses package their genomes. Our imaging and biophysical studies have revealed that the RSV Gag protein forms discrete nuclear, cytoplasmic, and plasma membrane foci that have properties of biological condensates, which have recently been shown to be important in regulating cell biology processes and virus-host interactions. We have observed that the Gag nuclear foci colocalize with unspliced viral RNA, suggesting that RSV Gag initially binds genomic RNA in the nucleus. In Aim 1, we will determine whether RSV Gag binds genomic RNAs at transcription sites using super-resolution live cell imaging, deep sequencing, single molecule fluorescence imaging, and CRISPR-based approaches. In Aim 2, we will use biophysical approaches to examine whether Gag-genomic RNA complexes form biological condensates that adopt properties of membrane-less organelles and undergo liquid-liquid phase separation. We will test the hypothesis that Gag-genomic RNA condensates remain tightly packed as they cross the nuclear envelope and traffic through the cytoplasm to the plasma membrane, where virus particles are assembled. Together, these experiments will move the field forward with new insights into how retroviruses select their RNA genomes and transport them to the plasma membrane for budding.

抽象的 逆转录病毒是正义单链 RNA 病毒，可导致癌症和严重疾病 动物和人类的免疫缺陷疾病，包括人类免疫缺陷病毒。对于超过一个 本世纪，引起家禽癌症的劳斯肉瘤病毒（RSV）已成为一种强大的模型 系统剖析逆转录病毒复制的分子基础，包括逆转录病毒组装。嘎嘎少校 逆转录病毒的结构蛋白，协调从血浆中出芽的病毒颗粒的组装 受感染细胞的膜。为了启动颗粒组装，Gag 选择性地结合未剪接的病毒 RNA 作为 病毒体中基因组RNA的来源。该提案重点关注 Gag 选择基因组的机制 RNA，解决该领域尚未解答的基本问题：(i) 细胞中的何处进行初始接触 Gag和病毒RNA之间发生； (ii) Gag如何选择性地招募未剪接的病毒RNA进行包装 当它仅占受感染细胞中总 RNA 的约 1% 时； (iii) Gag-viral 的特性是什么 促进通过细胞运输到质膜以释放颗粒的 RNA 复合物？因为 病毒颗粒从质膜中出芽，最初被认为是最初的 Gag 基因组 RNA 相互作用发生在细胞质中。我们的实验室发现 RSV Gag 进行核贩运， 这是有效的基因组病毒 RNA 包装所必需的。这一发现提出了 Gag 结合的可能性 细胞核中的基因组 RNA，这挑战了逆转录病毒如何包装其基因组的教条。 我们的成像和生物物理研究表明，RSV Gag 蛋白形成离散的核， 具有生物凝聚物特性的细胞质和质膜焦点，最近已被证实 已被证明在调节细胞生物学过程和病毒与宿主相互作用方面很重要。我们观察到 Gag 核灶与未剪接的病毒 RNA 共定位，表明 RSV Gag 最初结合基因组 细胞核中的RNA。在目标 1 中，我们将确定 RSV Gag 是否在转录位点结合基因组 RNA 使用超分辨率活细胞成像、深度测序、单分子荧光成像，以及 基于 CRISPR 的方法。在目标 2 中，我们将使用生物物理方法来检验 Gag-genomic 是否 RNA 复合物形成生物凝聚物，采用无膜细胞器的特性并经历 液-液相分离。我们将检验 Gag 基因组 RNA 凝聚物保持紧密的假设 当它们穿过核膜并通过细胞质到达质膜时，就会被包装起来，其中 病毒颗粒组装起来。总之，这些实验将通过新的见解推动该领域向前发展 逆转录病毒如何选择它们的 RNA 基因组并将其运输到质膜上进行出芽。