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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：
10441591
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
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/10441591
关键词：
Address Adopted Animals Automobile Driving Binding Biochemical Genetics 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 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.

摘要