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

基本信息

批准号：
10265719
负责人：
Susan R Weiss
金额：
$ 28.37万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10265719
关键词：
A549 Antiviral Agents Antiviral Response Apoptosis Binding Proteins CRISPR/Cas technology Cell Line Cell Nucleus Cells Cessation of life Chiroptera Cleaved cell 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 attenuation bat-borne betacoronavirus candidate identification design in vivo 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

项目摘要

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)- 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- 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.

中东呼吸综合征病毒 (MERS) 是 2012 年发现的人畜共患 C 型乙型冠状病毒除了SARS之外，MERS的出现还造成了2000多人感染和700多人死亡。强调了新出现的致命冠状病毒（MERS）对公共卫生的重要性。亲本蝙蝠冠状病毒 (BtCoV) 与其他蝙蝠传播的病毒一样，被认为其自然状态下是非致病性的蝙蝠和人类之间的人畜共患冠状病毒感染结果如此不同的原因在于。所有冠状病毒都编码谱系特异性辅助蛋白，通常在宿主拮抗作用中发挥作用。据报道，MERS 辅助蛋白 NS4a 和 NS4b 可以拮抗干扰素 (IFN)-。过度表达和报告系统的诱导，我们在此提供的数据显示任一突变然而，人们对此知之甚少。 MERS 感染期间宿主拮抗的机制是 NS4a 的另一个重要知识空白。是一种 dsRNA 结合蛋白，定位于病毒复制/转录复合物并拮抗 IFN- mRNA 表达，NS4b 与 NCBI 中的任何其他蛋白质没有同源性。鉴定 MERS NS4b 为 LigT 样 2H-磷酸酯酶 (2H-PE)，与 A 谱系的 NS2 蛋白相似 β冠状病毒 MHV，NS4b 具有 2',5'-磷酸二酯酶 (PDE) 活性，并拮抗 RNase L 然而，与 NS2 不同的是，MERS NS4b 具有 N 末端核定位信号 (NLS)，并且初步数据显示，NS4b 还可以切割可能存在的 RNA 中的 3',5' 键。底物，暗示其他可能的核功能表明 NS4b 调节抗病毒宿主。反应以及程序性细胞死亡途径，这可能至少部分是通过转录后我们将测试 MERS NS4a 和 NS4b 拮抗 dsRNA 的假设。 NS4b 是一种独特的冠状病毒蛋白，可在细胞质中诱导抗病毒途径在细胞核中酶促下调选定宿主 mRNA 的丰度，进一步拮抗抗病毒反应我们建议： 1. 使用重组 MERS 突变病毒来评估 NS4a。 NS4b介导的对人A549细胞和原代细胞中dsRNA诱导的抗病毒途径的拮抗作用 2.研究NS4b的底物特异性及其预测的核。在选定抗病毒 mRNA 丰度的转录后调节中的作用，并探索可能性 NS4b 调节程序性细胞死亡 3. 通过感染蝙蝠来识别蝙蝠特异性 MERS-宿主相互作用。这些研究将阐明带有 MERS 和 NS4a 和 NS4b 突变病毒的衍生细胞系和蝙蝠体内的情况。 MERS NS4a 和 NS4b 辅助蛋白可能具有多种功能，并长期导致此外，这些发现可能有助于解释高度的候选治疗靶点。与自然宿主相比，人畜共患病毒感染在人类中的致病结果。