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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.

摘要逆转录病毒是一种阳性的单链RNA病毒，会导致癌症和严重的动物和人类的免疫缺陷疾病，包括人类免疫缺陷病毒。加格，少校逆转录病毒的结构蛋白，协调从血浆中萌发的病毒颗粒的组装感染细胞的细胞膜。为了启动颗粒组装，GAG选择性地结合未剪接的病毒RNA作为病毒粒子中基因组RNA的来源。这一建议集中在Gag选择基因组的机制上。 RNA，解决领域中基本的、尚未回答的问题：(I)在细胞中的哪里进行初始接触在Gag和非剪接病毒RNA之间发生；(Ii)Gag如何选择性地招募非剪接病毒RNA用于仅占受感染细胞总RNA的~1%时进行包装；以及(Iii) GAG-病毒RNA复合体，促进通过细胞转运到质膜获得颗粒释放？因为病毒颗粒是从质膜上发芽的，所以最初认为- 基因组RNA的相互作用发生在细胞质中。我们实验室发现RSV Gag经历了核贩运，这是有效包装基因组病毒RNA所必需的。这一发现提高了 Gag与细胞核内的基因组RNA结合的可能性，挑战了逆转录病毒如何打包他们的基因组。我们的成像和生物物理研究表明，RSV Gag蛋白在具有生物分子凝聚体(BMC)特性的核、细胞质和质膜，它们已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。我们观察到Gag核焦点与未剪接的病毒RNA共存，表明RSV最初Gag 将基因组RNA结合在细胞核内。在这个资助项目的目标2中，我们正在使用生物物理方法来检查Gag-基因组RNA复合体是否表现出骨髓细胞的特性并经历液-液相分离。此外，我们正在验证Gag与细胞转录因子形成骨髓细胞的假设。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。这个本行政副刊《spectraMax®Paradigm®多模式》中要求提供的设备微型平板读取器和附带的墨盒将用于执行所述的生物物理实验目标2。该设备将允许我们执行最先进的、高通量的定量分析完成我们批准的实验计划的目标，并出版影响最大的手稿。