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

中东呼吸综合征病毒(MERS)是2012年发现的一种人畜共患病C系贝塔冠状病毒，已 导致2000多人感染，700多人死亡。非典之外的中东呼吸综合征的出现 强调新出现的强毒冠状病毒(CoV)对公共卫生的重要意义。MERS是由 亲代蝙蝠冠状病毒(BtCoV)和其他蝙蝠传播的病毒一样，被认为是天然的非致病性病毒 主持人。蝙蝠和人类之间人畜共患冠状病毒感染的结果如此不同的原因代表了 知识上的差距。所有冠状病毒都编码谱系特异的辅助蛋白，通常在宿主拮抗 先天反应。据报道，MERS辅助蛋白NS4a和NS4b可拮抗干扰素-β 在过表达和报告系统中诱导，我们在这里提供的数据显示其中一个的突变 蛋白质使重组突变型MERS病毒的复制减弱。然而，人们对此知之甚少。 MERS感染过程中宿主拮抗的机制，这是另一个重要的知识空白。而NS4a 是一种双链RNA结合蛋白，定位于病毒复制/转录复合体并拮抗干扰素-λ。 NS4B与NCBI中的其他蛋白没有同源性。我们使用结构建模来 鉴定MERS NS4B为LIGT样2H-磷酸酯酶(2H-PE)，与A系的NS2蛋白相似 β-冠状病毒MHV-NS4B具有2‘，5’-磷酸二酯酶活性并拮抗核糖核酸酶L 细胞质。然而，与NS2不同的是，MERS NS4B具有N端核定位信号(NLS)和 主要定位于细胞核。在初步数据中，NS4B还裂解了在可能的RNA中发现的3‘，5’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。 反应以及程序化的细胞死亡途径，这可能至少部分是通过转录后 修饰选定的mRNAs。我们将检验MERS NS4a和NS4B拮抗dsRNA- NS4B是一种独特的冠状病毒蛋白，它作用于 在细胞核中以酶的形式下调选择的宿主mRNAs的丰度，进一步 对抗抗病毒反应。我们建议：1.使用重组MERS突变病毒对NS4a进行评估 和NS4B介导的拮抗dsRNA诱导的人A549细胞和原代培养的抗病毒途径 人呼吸道上皮细胞。2.研究NS4B的底物特异性及其预测的核 在选择性抗病毒mRNAs丰度的转录后调节中的作用，并探索其可能性 NS4B调节程序性细胞死亡。3.通过感染BAT鉴定BAT特异的MERS-宿主相互作用 用MERS和NS4a、NS4B突变病毒体内衍生的细胞系和蝙蝠。这些研究将阐明 MERS NS4a和NS4B辅助蛋白可能具有多重功能，并在长期内导致 确定候选治疗靶点。此外，这些发现可能有助于解释 人畜共患病毒感染与其自然宿主相比的致病结局。

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.