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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

基本信息

批准号：
10797635
负责人：
Leslie J Parent
金额：
$ 10.71万
依托单位：
PENNSYLVANIA STATE UNIV HERSHEY MED CTR
依托单位国家：
美国
项目类别：
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/10797635
关键词：
Address Administrative Supplement Animals Automobile Driving Binding Biochemical Biological Biological Assay Biophysical Process Biophysics Cancer Etiology Cell Nucleus Cell membrane Cells Cellular biology Chromatin Complex Cytoplasm Cytoplasmic Granules Data Disease Equipment Exhibits Funding Genetic Transcription Genome Goals HIV Host Defense Human Immunologic Deficiency Syndromes Infection Integration Host Factors Intracellular Transport Knowledge Laboratories Liquid substance Manuscripts Measures Mediating Mediator Methods Modeling Molecular Morphogenesis Nuclear Nuclear Export Pathway interactions Pharmaceutical Preparations Phase Physical condensation Process Production Property Proteomics Publishing RNA RNA Binding RNA Polymerase II RNA Splicing RNA Viruses Reader Research Project Grants Retroviridae Retrovirus Proteins Ribonucleoproteins Role Rous sarcoma virus Site Source Structural Protein Structure Testing Thinking Travel Viral Virion Virus Assembly Work biophysical analysis biophysical properties biophysical techniques experimental study gag Gene Products genomic RNA imaging modality imaging study innovation microscopic imaging particle protein complex recruit trafficking transcription factor viral RNA viral genomics virus host interaction

项目摘要

Abstract Retroviruses are positive-sense, single-stranded RNA viruses that cause cancers and severe immunodeficiency diseases in animals and humans, including human immunodeficiency virus. 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 unspliced 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 foci in the nucleus, cytoplasm, and at the plasma membrane that have properties of biomolecular condensates (BMCs), which have 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 2 of this funded project, we are using biophysical approaches to examine whether Gag-genomic RNA complexes exhibit properties of BMCs and undergo liquid-liquid phase separation. In addition, we are testing the hypothesis that Gag forms BMCs with cellular transcription factors including Mediators and RNA polymerase II to facilitate genomic RNA packaging and virion assembly. The equipment being requested in this administrative supplement, the SpectraMax® Paradigm® Multi-Mode Microplate reader with accompanying cartridges, will be used to perform the biophysical experiments described in Aim 2. This equipment will allow us to perform state-of-the-art, high throughput, quantitative assays to accomplish the goals of our approved experimental plan and publish the highest impact manuscripts.

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。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和未剪接的病毒RNA之间发生； (ii) Gag 如何选择性地招募未剪接的病毒 RNA 当它仅包含受感染细胞中总 RNA 的约 1% 时进行包装； (iii) 的属性是什么 Gag-病毒 RNA 复合物促进颗粒通过细胞转运至质膜发布？由于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 经历了核运输，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 在细胞核中结合基因组 RNA 的可能性，这挑战了逆转录病毒如何结合的教条包装他们的基因组。我们的成像和生物物理研究表明，RSV Gag 蛋白在细胞核、细胞质和质膜具有生物分子凝聚物 (BMC) 的特性，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 最初结合细胞核中的基因组 RNA。在这个资助项目的目标 2 中，我们正在使用生物物理方法检查 Gag-基因组 RNA 复合物是否表现出 BMC 的特性并经历液-液相分离。此外，我们正在测试 Gag 与细胞转录因子形成 BMC 的假设包括介体和 RNA 聚合酶 II，以促进基因组 RNA 包装和病毒粒子组装。这本行政补充中要求的设备是 SpectraMax® Paradigm® 多模式带有随附卡盒的酶标仪将用于执行所述的生物物理实验目标 2。该设备将使我们能够进行最先进的、高通量的定量分析，以完成我们批准的实验计划的目标并发表最具影响力的手稿。