MERS coronavirus: antagonism of double-stranded RNA induced host response by accessory proteins

MERS 冠状病毒：辅助蛋白拮抗双链 RNA 诱导的宿主反应

• 批准号: 10396471
• 负责人: Susan R Weiss
• 金额: $ 44.76万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-24 至 2023-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10396471
• 关键词: A549; Antiviral Response; Apoptosis; Binding Proteins; CRISPR/Cas technology; Cell Line; Cell Nucleus; Cells; Cessation of life; Chiroptera; Complement; Complex; Coronavirus; Coronavirus Infections; Cytoplasm; Data; Detection; Disease; Double-Stranded RNA; Engineering; Enzymes; Epithelial; Epithelial Cells; Genes; Genetic Transcription; Human; Immune response; Infection; Innate Immune Response; Interferon-beta; Interferons; Knock-out; Knowledge; Lead; Ligands; Lung infections; Mediating; Messenger RNA; Middle East Respiratory Syndrome Coronavirus; Modification; Murine hepatitis virus; Mutation; N-terminal; Nuclear; Nuclear Localization Signal; Outcome; Pathogenicity; Pathway interactions; Post-Transcriptional Regulation; Proteins; Public Health; Publishing; RNA; Recombinants; Reporter; Reporting; Ribonucleases; Role; Severe Acute Respiratory Syndrome; Structural Models; Substrate Specificity; System; Testing; Therapeutic; Viral; Virulence Factors; Virulent; Virus; Virus Diseases; Virus Receptors; Virus Replication; Zoonoses; airway epithelium; antagonist; attenuation; bat-borne; betacoronavirus; candidate identification; design; in vivo; interferon antagonist; mRNA Expression; mutant; novel coronavirus; oligoadenylate; overexpression; phosphodiesterase V; phosphoric diester hydrolase; recombinant virus; replicase; response; sensor; therapeutic target; transcriptome sequencing; transmission process; virus host interaction; zoonotic coronavirus

Middle East respiratory syndrome virus (MERS), a zoonotic lineage C Betacoronavirus discovered in 2012, has caused over 2,000 infections and more than 700 deaths. The emergence of MERS in addition to SARS highlights the public health significance of virulent, emerging coronaviruses (CoVs). MERS is descended from a parental bat CoV (BtCoV), and like other bat borne viruses, is believed to be nonpathogenic in its natural host. The reasons for such disparate outcomes of zoonotic CoV infection between bats and humans represent a gap in knowledge. All CoVs encode lineage specific accessory proteins often with roles in host antagonism of innate responses. MERS accessory proteins NS4a and NS4b are reported to antagonize interferon (IFN)-β induction in overexpression and reporter systems, and we present data herein showing that mutation of either protein confers attenuation of replication to recombinant mutant MERS viruses. However, little is known about the mechanisms of host antagonism during MERS infection, another important gap in knowledge. While NS4a is a dsRNA binding protein that localizes with viral replication/transcription complexes and antagonizes IFN-λ mRNA expression, NS4b has no homology with any other protein in NCBI. We used structural modeling to identify MERS NS4b as a LigT-like 2H-phosphoesterases (2H-PE), and like the NS2 protein of lineage A Betacoronavirus MHV, NS4b has 2’,5'-phosphodiesterase (PDE) activity and antagonizes RNase L in the cytoplasm. However, unlike NS2, MERS NS4b has an N-terminal nuclear localization signal (NLS) and localizes primarily to the nucleus. In preliminary data, NS4b also cleaves 3’,5’ bonds found in possible RNA substrates, implying other likely nuclear functions. RNA-seq data suggest that NS4b regulates the antiviral host responses as well as programmed cell death pathways, and this may be at least in part by post-transcriptional modification of select mRNAs. We will test the hypothesis that MERS NS4a and NS4b antagonize dsRNA- induced antiviral pathways in the cytoplasm and NS4b is a unique coronavirus protein, which acts enzymatically in the nucleus to down-regulate the abundance of select host mRNAs, further antagonizing antiviral responses. We propose to: 1. Use recombinant MERS mutant viruses to assess NS4a and NS4b-mediated antagonism of dsRNA-induced antiviral pathways in human A549 cells and in primary human airway epithelial cells. 2. Investigate the substrate specificity of NS4b as well as its predicted nuclear role in post-transcriptional regulation of the abundance of select antiviral mRNAs, and explore the possibility that NS4b modulates programmed cell death. 3. Identify bat specific MERS-host interactions by infection of bat derived cell lines and bats in vivo with MERS and NS4a and NS4b mutant viruses. These studies will elucidate the likely multiple functions of the MERS NS4a and NS4b accessory proteins and in the long-term lead to identification of candidate therapeutic targets. In addition, these findings may help explain the highly pathogenic outcome of zoonotic virus infection in humans as compared to their natural hosts.

中东呼吸道综合症病毒（MERS）是2012年发现的人畜共患病C 引起了2,000多次感染和700多人死亡。除了SARS之外，MER的出现 强调了病毒，新兴冠状病毒（COVS）的公共健康意义。 MERS从 父母蝙蝠COV（BTCOV）和其他蝙蝠出生的病毒与其他蝙蝠一样，自然而然地是非病毒的 主持人。蝙蝠与人之间的人畜共患性COV感染的这种不同结果的原因代表 知识差距。所有COV都经常在宿主对拮抗作用中编码特定谱系蛋白 天生的回应。据报道，MERS辅助蛋白NS4A和NS4B拮抗干扰素（IFN）-β 过表达和报告基因系统的诱导，我们在此介绍数据，显示了这两种突变 蛋白质承认复制对重组突变MERS病毒的衰减。但是，对 MERS感染期间宿主拮抗作用的机制，这是知识的另一个重要差距。而NS4A 是DSRNA结合蛋白，它以病毒复制/转录复合物定位并拮抗IFN-λ mRNA表达，NS4B与NCBI中的任何其他蛋白质都没有同源性。我们将结构建模用于 将MERS NS4B识别为LIGT样的2H磷酸酯酶（2H-PE），并且像谱系A的NS2蛋白一样 betacoronavirus MHV，NS4B具有2'，5'-磷酸二酯酶（PDE）活性，并拮抗RNase L 细胞质。但是，与NS2不同，MERS NS4B具有N末端核定位信号（NLS）和 主要定位于细胞核。在初步数据中，NS4B还裂解了3'，5'在可能的RNA中发现的键 底物，暗示其他可能的核功能。 RNA-seq数据表明NS4B调节抗病毒宿主 响应以及编程的细胞死亡途径，至少部分是由转录后的 修改精选mRNA。我们将检验以下假设：MERS NS4A和NS4B拮抗dsRNA- 细胞质和NS4B中诱导的抗病毒途径是独特的冠状病毒蛋白，它起作用 在核中酶促下调选择宿主mRNA的抽象，进一步 拮抗抗病毒反应。我们建议：1。使用重组MERS突变病毒评估NS4A NS4B介导的DSRNA诱导的抗病毒途径在人A549细胞中以及原发性的拮抗剂 人气道上皮细胞。 2。研究NS4B及其预测的核的底物特异性 在选定的选拔抗病毒mRNA抽象的转录后调节中的作用，并探索可能性 NS4B调节编程的细胞死亡。 3。通过感染BAT确定BAT特定的MERS-HOST相互作用 带有MERS和NS4A和NS4B突变病毒的体内细胞系和蝙蝠。这些研究将阐明 MERS NS4A和NS4B辅助蛋白的可能多个功能，并且长期导致 识别候选治疗靶标。此外，这些发现可能有助于高度解释 与自然宿主相比，人畜共患病毒感染的致病结果。