Molecular Mechanisms of Retroviral Gag-RNA interactions in Virus Assembly

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。了一个多 世纪以来，引起家禽癌症的劳斯肉瘤病毒（Rous sarcoma virus，RSV 系统剖析逆转录病毒复制的分子基础，包括逆转录病毒装配。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和病毒RNA之间发生;（ii）Gag如何选择性地招募未剪接的病毒RNA进行包装 当它仅占感染细胞总RNA的~1%时;以及（iii）GAG病毒的特性是什么 RNA复合物，促进运输通过细胞到质膜的颗粒释放？因为 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经历了核贩运， 这是有效的基因组病毒RNA包装所必需的。这一发现增加了Gag结合 基因组RNA在细胞核中，这挑战了逆转录病毒如何包装其基因组的教条。 我们的成像和生物物理学研究揭示了RSV Gag蛋白形成离散的核， 细胞质和质膜病灶，具有生物凝聚物的性质，最近 已经证明在调节细胞生物学过程和病毒-宿主相互作用中是重要的。我们观察到 Gag核灶与未剪接的病毒RNA共定位，表明RSV Gag最初结合基因组 核中的RNA。在目标1中，我们将确定RSV Gag是否在转录位点结合基因组RNA 使用超分辨率活细胞成像，深度测序，单分子荧光成像， 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是否存在GAG-基因组 RNA复合物形成生物浓缩物，其采用无膜细胞器的特性， 液-液相分离我们将检验这样的假设，即GAG-基因组RNA浓缩物保持紧密 当它们穿过核膜并穿过细胞质到达质膜时， 病毒颗粒被组装。这些实验将共同推动该领域的发展， 逆转录病毒如何选择它们的RNA基因组并将其转运到质膜上进行出芽。

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.