Perturbation of Cellular Translation and RNA Metabolism by SARS-COV-2 Nucleoprotein Phase Separation

本研究将探讨严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）N蛋白相分离在干扰细胞翻译和RNA代谢中的作用。N蛋白相分离产生无膜细胞器，并将RNA浓缩在液滴（致密）相中。虽然N蛋白负责病毒基因组的降解，但N蛋白也结合数百种宿主信使RNA，其中一些是翻译和无义介导的衰变（NMD）所必需的。NMD靶向RNA衰变的RNA病毒的不同家族，包括β冠状病毒，并且来自鼠肝炎病毒（MHV）的N蛋白在感染的早期阶段干扰NMD。由于NMD靶向10%的细胞转录物进行降解，N蛋白的抑制可能对宿主细胞转录组具有深远的影响。虽然N蛋白抑制NMD的机制尚不清楚，但相分离和N蛋白的翻译抑制可能是一个主要因素。已知细胞蛋白FUS和C9 orf 72富含精氨酸的二肽重复序列相分离、抑制翻译、抑制NMD，并且与肌萎缩性侧索硬化症（ALS）疾病进展相关。病毒蛋白，包括N蛋白，可以抑制共翻译NMD途径所必需的转录物的翻译，潜在地以有利于病毒复制的方式塑造宿主转录组。最近的研究表明，SARS-CoV-2感染诱导炎性细胞因子的高表达，但未能建立一个强大的干扰素反应。驱动这些转录反应的潜在因素仍然未知，然而，N蛋白可以通过干扰NMD和阻断转录后调控来引发这种反应。该项目将使用全转录组核糖体分析（Ribo-seq）来识别被N蛋白抑制的细胞转录物。将检查先前鉴定的NMD靶标以确定翻译抑制是否赋予NMD抗性。将平行表达相分离缺陷型N突变体，以确定相分离是否是翻译和NMD抑制的驱动力。最后，本研究产生的结果将与已发表的SARS-CoV-2感染细胞的RNA-seq数据集交叉引用，以确定N蛋白在感染期间对基因表达大规模变化的贡献程度。这些发现将使我们更好地了解SARS-CoV-2感染期间发生的病毒-宿主相互作用，并将支持正在进行的疫苗和抗病毒药物开发，特别是基于N的方法。

This proposal will investigate the role severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N protein phase separation in disrupting cellular translation and RNA metabolism. N protein phase separation creates membraneless organelles and concentrates RNAs in a droplet (dense) phase. While N protein is responsible for encapsidation of the viral genome, N protein also binds hundreds of host messenger RNAs, some of which are necessary for translation and nonsense-mediated decay (NMD). NMD targets diverse families of RNA viruses for RNA decay, including betacoronaviruses, and the N protein from Murine hepatitis virus (MHV) interferes with NMD during the early stages of infection. Since NMD targets 10% of cellular transcripts for degradation, inhibition by N protein likely has profound effects on the host cell transcriptome. While the mechanism underlying the inhibition of NMD by N protein remains unclear, phase separation and translational repression by N could be a predominant factor. The cellular proteins FUS and C9orf72 arginine- rich dipeptide repeats are known to phase separate, repress translation, inhibit NMD, and are associated with Amyotrophic lateral sclerosis (ALS) disease progression. Viral proteins, including N protein, may repress the translation of transcripts necessary for the co-translational NMD pathway, potentially shaping the host transcriptome in a way that favors virus replication. Recent work has shown that SARS-CoV-2 infection induces high expression of inflammatory cytokines but fails to mount a robust interferon response. The underlying factors driving these transcriptional responses remain unknown; however, N protein could provoke this response by interfering with NMD and blocking post-transcriptional regulation. This project will use transcriptome-wide ribosome profiling (Ribo-seq) to identify cellular transcripts that are translationally repressed by N protein. Previously identified NMD targets will be examined to determine if translational repression confers NMD-resistance. Phase-separation deficient N mutants will be expressed in parallel to determine whether phase separation is the driving force in translation and NMD inhibition. Finally, the results generated from this study will be cross-referenced with published RNA-seq datasets from SARS-CoV-2 infected cells to determine the extent that N protein contributes to the large-scale changes in gene expression during infection. These findings will better our understanding of the virus-host interactions that take place during SARS-CoV-2 infection and will support on-going vaccine and antiviral development, especially N-based approaches.