Molecular Mechanisms of Retroviral Gag-RNA interactions in Virus Assembly

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。已经超过一年了 世纪之交，引起家禽癌症的劳斯肉瘤病毒(RSV)已成为一种强有力的模型 系统剖析逆转录病毒复制的分子基础，包括逆转录病毒组装。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 Gag与病毒RNA之间的关系；(Ii)Gag如何选择性地招募未剪接的病毒RNA进行包装 当它只占受感染细胞总RNA的1%时；以及(Iii)Gag病毒的特性是什么 促进通过细胞转运到质膜以释放颗粒的RNA复合体？因为 病毒颗粒从质膜上发芽，最初被认为是搞笑基因组的RNA 相互作用发生在细胞质中。我们的实验室发现RSV Gag经历了核贩运， 这是有效包装基因组病毒RNA所必需的。这一发现增加了GAG结合的可能性 细胞核中的基因组RNA，这对逆转录病毒如何包装其基因组的教条提出了挑战。 我们的成像和生物物理研究表明，RSV Gag蛋白形成离散的核， 具有生物凝集物特性的细胞质和质膜病灶，最近 已被证明在调节细胞生物学过程和病毒与宿主的相互作用方面具有重要作用。我们观察到 Gag核焦点与未剪接的病毒RNA共存，表明RSV Gag最初与基因组结合 核内有核糖核酸。在目标1中，我们将确定rsv gag是否在转录位点与基因组rna结合。 使用超分辨率活细胞成像，深度测序，单分子荧光成像，以及 基于CRISPR的方法。在目标2中，我们将使用生物物理方法来检查Gag基因组 RNA复合体形成生物凝聚体，具有无膜细胞器的特性，并经历 液-液相分离。我们将检验这样一种假设，即插嘴基因组rna凝聚物保持紧密。 当它们穿过核膜并通过细胞质运输到质膜时被包装起来，在那里 病毒颗粒被组装在一起。总之，这些实验将以新的见解推动该领域向前发展 逆转录病毒如何选择它们的RNA基因组，并将它们运送到质膜上发芽。

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.