Coronavirus Antagonism of the OAS-RNase L Pathway

冠状病毒对 OAS-RNase L 途径的拮抗作用

• 批准号: 8908529
• 负责人: Joshua Mathew Thornbrough
• 金额: $ 4.65万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2015-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8908529
• 关键词: Acute Hepatitis; Adult Respiratory Distress Syndrome; Affect; Alveolar Macrophages; Antiviral Agents; Antiviral Response; Apoptosis; Biological Assay; Birds; Bone Marrow; Cells; Chiroptera; Cleaved cell; Common Cold; Coronavirus; Data; Development; Disease; Double-Stranded RNA; Endothelial Cells; Epithelial Cells; Figs - dietary; Future; Genes; Hepatitis; Hepatocyte; High Pressure Liquid Chromatography; Homologous Gene; Homologous Protein; Human; Human Coronavirus 229E; Human Coronavirus OC43; Immune; In Vitro; Infection; Interferon Type I; Interferons; Kupffer Cells; Lead; Life; Ligase; Link; Liver; Lung; Lung diseases; Measures; Mediating; Messenger RNA; Middle East Respiratory Syndrome Coronavirus; Murine hepatitis virus; Mus; Natural Immunity; Pathogenesis; Pathway interactions; Play; Poly I-C; Protein Biosynthesis; Proteins; RNA Degradation; Recombinants; Reporting; Respiratory System; Respiratory Tract Infections; Respiratory tract structure; Ribonucleases; Ribosomal RNA; Role; SARS coronavirus; Severe Acute Respiratory Syndrome; Signal Transduction; Structure; Testing; Therapeutic; Therapeutic Effect; Tropism; Viral; Viral Genome; Viral Pathogenesis; Virulence; Virus; Virus Replication; base; cell type; human coronavirus; liver infection; macrophage; member; mortality; mutant; oligoadenylate; overexpression; pathogen; phosphodiesterase V; phosphoric diester hydrolase; pneumocyte; prevent; public health relevance; respiratory; response; small molecule; success; treatment strategy; virus host interaction

 DESCRIPTION (provided by applicant): Coronaviruses cause a broad spectrum of respiratory disease in humans. Human coronaviruses (HCoV)-OC43 and HCoV-229E have long been known as agents of the common cold. The emergence of SARS-CoV as the etiological agent of severe acute respiratory distress syndrome in 2003 and in 2012, Middle East respiratory syndrome coronavirus (MERS-CoV), with 10% and 30% mortality, respectively, make it imperative to understand the coronavirus-host interactions that contribute to virulence. The ability of viruses to evade or antagonize type I interferon (IFN) signaling, influences viral pathogenesis. An important, but understudied aspect of IFN evasion by coronaviruses is antagonism of the potent, antiviral oligoadenylate synthetase (OAS)- ribonuclease (RNase) L pathway. Once activated by double stranded RNA, (dsRNA), OAS synthesizes 2',5'- linked oligoadenylates (2-5A) that activate RNase L. RNase L cleaves single stranded RNA leading to degradation of viral genomes, arrest of protein synthesis, and apoptosis. The group 2a Betacoronavirus, mouse hepatitis virus (MHV) accessory protein ns2 is a 2',5'-phosphodiesterase (PDE) that cleaves 2-5A thereby preventing RNase L activation. PDE activity is a critical determinant of MHV hepatovirulence in mice. Other group 2a Betacoronavirus, including HCoV-OC43, encode ns2 homologs recently confirmed as RNase L antagonists. Additionally, ORF4b of the group 2c Betacoronavirus MERS-CoV, encodes a protein predicted to have PDE activity. ORF4b homologs are encoded by all sequenced group 2c Betacoronavirus. These findings lead to the overall hypothesis that OAS-RNase L antagonism contributes to human group 2a Betacoronavirus respiratory pathogenesis and that group 2c Betacoronavirus also antagonize the OAS-RNase L pathway by PDE mediated cleavage of 2-5A, contributing to virulence. I will carry out the following aims. Aim 1 will examin the contribution of RNase L antagonism to the success of HCoV-OC43 in primary human airway cells. Metrics for analysis of antagonism will include viral titers, ribosomal RNA (rRNA) integrity and quantification of 2-5A levels. Results will be correlated with expression of OAS genes in these cells. Aim 2 will investigate the predicted PDE activity of MERS-CoV encoded ORF4b protein and two Bat-CoV homologs. PDE activity of recombinant ORF4b encoded protein will be assessed in vitro by a 2-5A cleavage assay and in transfected cells by measuring rRNA integrity and quantifying 2-5A levels by an RNase L activation assay. In addition, chimeric MHVs expressing inactive ns2 and each active ORF4b PDE will be constructed, infections performed in mice and rescue of replication and hepatitis assessed. Finally, primary human airway cells will be infected with MERS-CoV or ORF4b mutant virus to assess replication in relevant human cells. This study will contribute to the understanding of coronavirus-host interactions, in particular the importance of RNase L antagonism by human respiratory coronaviruses. Finally, viral PDEs offer a tantalizing target for antiviral therapeutics by isolating treatment to infected cells, because viral dsRNA is required for RNase L activation.

 描述（由适用提供）：冠状病毒引起人类的各种呼吸道疾病。人类冠状病毒（HCOV）-OC43和HCOV-229E长期以来被称为普通感冒的药物。 SARS-COV作为2003年严重急性呼吸窘迫综合征的病因学药物的出现，2012年，中东呼吸综合征冠状病毒（MERS-COV）分别具有10％和30％的死亡率，因此必须理解有助于病毒的冠状病毒 - 霍斯特 - 霍斯特 - 霍斯特 - 霍斯特 - 霍斯特 - 霍斯特 - 霍斯特 - 霍斯特 - 霍斯特 - 霍斯特 - 霍斯特 - 霍斯特 - 霍斯特 - 霍斯特相互作用。病毒逃避或拮抗I型干扰素（IFN）信号传导的能力，会影响病毒发病机理。冠状病毒进化的一个重要但知识的方面是抗病毒寡腺苷酸合成酶（OAS） - 核糖核酸酶（RNase）L途径的拮抗作用。一旦被双链RNA（dsRNA）激活，OAS合成了2'，5'-链接的寡腺苷酸（2-5a），该烯基（2-5a）激活了RNase L.RNase L.RNase L裂解的单链RNA，导致病毒基因组降解，蛋白质合成和凋亡。第2A组Betacoronavirus，小鼠肝炎病毒（MHV）辅助蛋白NS2是2'，5'-磷酸二酯酶（PDE），从而裂解2-5A，从而防止RNase L激活。 PDE活性是小鼠MHV肝病病毒的关键决定性决定。包括HCOV-OC43在内的其他2A组BETACORONAVIRUS最近证实NS2同源物是RNase L拮抗剂。另外，2C组的ORF4B betacoronavirus mers-cov编码预测具有PDE活性的蛋白质。 ORF4B同源物由所有测序组2C Betacoronavirus编码。这些发现导致了总体假设，即OAS-RNase L拮抗作用有助于人类2A组BETACORONAVIRUS呼吸发病机理，并且2C组Betacoronavirus组也通过PDE介导的2-5A清洁剂拮抗OAS-RNase L途径，从而有助于病毒。我将执行以下目标。 AIM 1将研究RNase L拮抗作用对HCOV-OC43在原代人气道细胞中成功的贡献。用于分析拮抗的指标将包括病毒滴度，核糖体RNA（rRNA）完整性 和2-5A水平的定量。结果将与这些细胞中OAS基因的表达相关。 AIM 2将研究MERS-COV编码的ORF4B蛋白和两个BAT-COV同源物的PDE活性。重组ORF4B编码蛋白的PDE活性将在体外通过2-5A裂解测定法评估，并在翻译细胞中通过测量RRNA完整性并通过RNase L活性测定法对2-5A水平进行量化。此外，将构建表达无活性NS2和每个主动ORF4B PDE的嵌合MHV，在小鼠中进行感染，并评估复制和肝炎的救助。最后，原发性人类气道细胞将被MERS-COV或ORF4B突变病毒感染，以评估相关人类细胞中的复制。这项研究将有助于理解冠状病毒 - 宿主相互作用，特别是人类呼吸道冠状病毒对RNase L拮抗作用的重要性。最后，病毒PDE通过将治疗隔离为感染，为抗病毒治疗提供了诱人的靶标 细胞，因为RNase L活化需要病毒DSRNA。