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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.

摘要逆转录病毒是正义单链RNA病毒，可导致癌症和严重的癌症。在动物和人类中的免疫缺陷疾病，包括人类免疫缺陷病毒。加格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装配感染细胞的膜。为了启动颗粒组装，Gag选择性地结合未剪接的病毒RNA，因为Gag是一种非剪接的RNA。病毒体中基因组RNA的来源。该建议侧重于Gag选择基因组的机制， RNA，解决基本的，在该领域未回答的问题：（i）在细胞中的初始接触 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，当它只占感染细胞中总RNA的~1%时，包装;以及（iii）促进颗粒通过细胞转运至质膜的GAG-病毒RNA复合物释放？由于病毒颗粒从质膜出芽，最初认为最初的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 核运输，这是有效的基因组病毒RNA包装所必需的。这一发现提高了 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包装它们的基因组。我们的成像和生物物理学研究已经揭示了RSV Gag蛋白在细胞中形成离散的病灶。细胞核、细胞质和质膜具有生物分子凝聚物（BMC）的性质，其在调节细胞生物学过程和病毒-宿主相互作用中显示出重要性。我们我观察到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。在该资助项目的目标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的性质并经历液-液相分居此外，我们正在验证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包装和病毒体组装。的本管理补充中要求的设备，SpectraMax® Paradigm®多模式酶标仪和随附试剂盒将用于进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，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。