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(申请人提供)：冠状病毒可引起人类多种呼吸道疾病。人类冠状病毒(HCoV)-OC43和HCoV-229E长期以来一直被认为是普通感冒的病原体。2003年SARS冠状病毒作为严重急性呼吸窘迫综合征的病原体出现，2012年中东呼吸综合征冠状病毒(MERS-CoV)的死亡率分别为10%和30%，这使得了解冠状病毒与宿主之间的相互作用成为当务之急。病毒逃避或拮抗I型干扰素信号的能力影响病毒的致病机制。冠状病毒逃避干扰素的一个重要方面是对有效的抗病毒寡腺苷合成酶-核糖核酸酶L通路的拮抗作用。一旦被双链核糖核酸(DsRNA)激活，OAS就会合成2‘，5’-连接的寡腺苷(2-5A)，激活核糖核酸酶L。核糖核酸酶L裂解单链核糖核酸，导致病毒基因组降解、蛋白质合成受阻和细胞凋亡。2a组β冠状病毒，小鼠肝炎病毒辅助蛋白NS2是一种2‘，5’-磷酸二酯酶，它能裂解2-5A，从而阻止核糖核酸酶L的激活。PDE活性是MHV对小鼠肝脏毒力的关键决定因素。另一组2aβ冠状病毒，包括人类冠状病毒OC43，编码最近被证实为核糖核酸酶L拮抗剂的NS2同源物。此外，2c组BetacoronaVirus MERS-CoV的ORF4b编码一种预测具有PDE活性的蛋白质。ORF4b同源物由所有测序的2c组BetacoronaVirus编码。这些发现导致了一个总体假设，即OAS-核糖核酸酶L拮抗作用与人类2a组贝塔冠状病毒的呼吸道致病有关，而2c组贝塔冠状病毒也通过PDE介导的2-5A的裂解而拮抗OAS-核糖核酸酶L途径，从而有助于毒力的产生。我将实现以下目标。目的1研究核糖核酸酶L拮抗剂对人冠状病毒OC43在原代人呼吸道细胞感染中的作用。分析拮抗作用的指标将包括病毒滴度、核糖体RNA(RRNA)完整性 以及2-5A水平的量化。结果将与这些细胞中OAS基因的表达相关。目的研究MERS冠状病毒编码的ORF4b蛋白和两个BAT冠状病毒同源蛋白的PDE活性。重组ORF4b编码蛋白的PDE活性将在体外通过2-5A裂解试验进行评估，并将在转基因细胞中通过测量rRNA完整性和通过核糖核酸酶L激活试验来定量2-5A水平。此外，还将构建表达非活性NS2和每个活性ORF4b PDE的嵌合MHV，在小鼠身上进行感染，并评估复制和肝炎的抢救。最后，原代人类呼吸道细胞将被MERS-CoV或ORF4b突变病毒感染，以评估相关人类细胞的复制情况。本研究将有助于了解冠状病毒与宿主的相互作用，特别是人类呼吸道冠状病毒对核糖核酸酶L拮抗作用的重要性。最后，病毒PDE通过隔离感染的治疗为抗病毒治疗提供了一个诱人的靶点。 细胞，因为病毒dsRNA是激活核糖核酸酶L所必需的。