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：
10241537
负责人：
Leslie J Parent
金额：
$ 42.04万
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 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10241537
关键词：
Address Adopted Animals Automobile Driving Binding Biochemical Genetics Biological Biological Models Biophysical Process Biophysics Cancer Etiology Cell Nucleus Cell membrane Cells Cellular biology Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Complex Cytoplasm Cytoplasmic Granules DNA Data Disease Domestic Fowls 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 Play Process Production Property Proteomics Publishing RNA RNA Binding RNA Splicing RNA Viruses Research Project Grants Resolution Retroviridae Retrovirus Proteins Ribonucleoproteins Role Rous sarcoma virus Site Source Structural Protein Structure Testing Thinking Transcript Travel Viral Virion Virus Assembly Work base 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 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. 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阳性，单链的RNA病毒，引起癌症和严重动物和人类的免疫缺陷疾病，包括人类免疫缺陷病毒。超过一个 Century，在国内禽类中引起癌症的Rous肉瘤病毒（RSV）一直是一个强大的模型剖析逆转录病毒复制的分子基础的系统，包括逆转录病毒组件。插科打，专业逆转录病毒的结构蛋白质策划了从血浆中芽芽的病毒颗粒组装感染细胞的膜。为了启动颗粒组件，插科打g选择性地结合未斑型病毒RNA作为病毒体中基因组RNA的来源。该提案重点介绍了插科打gag选择基因组的机制 RNA，在现场解决基本的，未解决的问题：（i）在单元中进行初始接触的位置在插科打g和病毒RNA之间发生；（ii）GAG如何选择性地募集未填充的病毒RNA用于包装当它仅占感染细胞中总RNA的约1％时；（iii）GAG病毒的特性是什么 RNA复合物可促进通过细胞传输到质膜以释放质膜？因为病毒颗粒来自质膜的芽，最初认为初始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插科打经过核贩运，这是有效的基因组病毒RNA包装所必需的。这一发现提出了堵嘴绑定的可能性细胞核中的基因组RNA，这挑战了逆转录病毒打包其基因组的教条。我们的成像和生物物理研究表明，RSV GAG蛋白形成离散的核，具有生物冷凝物特性的细胞质和质膜灶被证明在调节细胞生物学过程和病毒宿主相互作用方面很重要。我们已经观察到与未填充的病毒RNA共定位的GAG核灶，这表明RSV GAG最初结合基因组核中的RNA。在AIM 1中，我们将确定RSV GAG是否在转录位点结合基因组RNA 使用超分辨率的活细胞成像，深度测序，单分子荧光成像和基于CRISPR的方法。在AIM 2中，我们将使用生物物理方法来检查GAG基因组是否 RNA复合物形成生物冷凝物，采用无膜细胞器的特性并经历液 - 液相分离。我们将检验以下假设：GAG基因组RNA冷凝物保持紧密当它们越过核包膜和通过细胞质交通交通时，它们被包装到质膜，在那里病毒颗粒组装。这些实验将共同将领域的新见解推向逆转录病毒如何选择其RNA基因组并将其运输到质膜以进行萌芽。