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

SARS-COV-2 核蛋白相分离对细胞翻译和 RNA 代谢的干扰

基本信息

批准号：
10516080
负责人：
Jared Paul May
金额：
$ 7.83万
依托单位：
UNIVERSITY OF MISSOURI KANSAS CITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-11-01 至 2023-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10516080
关键词：
2019-nCoV Amyotrophic Lateral Sclerosis Arginine Automobile Driving Basic Science Binding C-terminal C9ORF72 COVID-19 COVID-19 pandemic COVID-19 patient Cells Cessation of life Coronavirus Data Set Development Dipeptides Disease Disease Outbreaks Disease Progression Event Family Future G3BP1 gene Gene Expression Genetic Transcription Green Fluorescent Proteins Hela Cells High-Throughput Nucleotide Sequencing Immunoassay Infection Inflammatory Interferons Length Life Cycle Stages Mediating Membrane Messenger RNA Middle East Respiratory Syndrome Coronavirus Murine hepatitis virus N Domain Nature Nonsense-Mediated Decay Nucleoproteins Organelles Pathway interactions Phase Phosphorylation Post-Transcriptional Regulation Process Proteins Publishing RNA RNA Decay RNA Degradation RNA Viruses RNA metabolism RNA-Binding Protein FUS Reporter Repression Resistance Ribosomal RNA Role SARS coronavirus SARS-CoV-2 infection Shapes Therapeutic Transcript Translating Translational Repression Translations United States Vaccines Variant Viral Genome Viral Interference Viral Proteins Virus Virus Replication Work antiviral drug development betacoronavirus cytokine design differential expression driving force effective therapy improved mRNA Stability mutant protein expression response ribosome profiling stress granule transcriptome transcriptome sequencing vaccine candidate viral RNA virus host interaction

项目摘要

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.

该建议将研究严重急性呼吸综合症冠状病毒2（SARS-COV-2）N蛋白相分离在破坏细胞翻译和RNA代谢中的作用。 N蛋白相分离会产生无膜细胞器，并在液滴（致密）相中浓缩RNA。尽管N蛋白负责封装病毒基因组，但N蛋白也结合了数百个宿主Messenger RNA，其中一些是翻译和胡说八道介导的衰减（NMD）所必需的。 NMD靶向RNA病毒的各种家族用于RNA衰减，包括betacoronaviruses，以及在感染的早期阶段，来自鼠肝炎病毒（MHV）的N蛋白会干扰NMD。由于NMD靶向10％的细胞转录本以降解，因此N蛋白的抑制作用可能对宿主细胞转录组具有深远的影响。尽管N蛋白对NMD抑制的基础机制尚不清楚，但N相分离和n的平移抑制可能是主要因素。已知细胞蛋白FUS和C9ORF72精氨酸 - 富二肽重复序列可以分开，抑制翻译，抑制NMD，并与肌萎缩性侧面硬化症（ALS）疾病进展有关。病毒蛋白（包括N蛋白）可能会抑制共同翻译NMD途径所需的转录本的翻译，从而可能以有利于病毒复制的方式塑造宿主转录组。最近的工作表明，SARS-COV-2感染诱导炎症细胞因子的高表达，但无法安装强大的干扰素反应。推动这些转录反应的基本因素仍然未知；但是，N蛋白可以通过干扰NMD并阻止转录后调节来引起这种反应。该项目将使用整个转录组核糖体分析（Ribo-Seq）来识别N蛋白翻译抑制的细胞转录本。将检查先前确定的NMD目标，以确定翻译抑制是否赋予NMD抗性。相分离不足的n突变体将平行表达，以确定相位分离是否是翻译和NMD抑制的驱动力。最后，这项研究产生的结果将与来自SARS-COV-2感染细胞的RNA-Seq数据集进行交叉引用，以确定N蛋白有助于感染过程中基因表达的大规模变化的程度。这些发现将更好地理解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发育，尤其是基于N的方法。