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C系β冠状病毒， 造成了两千多人感染，七百多人死亡除了SARS，MERS的出现 强调了新出现的致命冠状病毒（CoV）的公共卫生意义。MERS起源于 亲本蝙蝠冠状病毒（BtCoV）和其他蝙蝠携带的病毒一样，被认为在其自然状态下是非致病性的。 主持人蝙蝠和人类之间的人畜共患病CoV感染结果如此不同的原因代表了 知识上的差距。所有CoV都编码谱系特异性辅助蛋白，通常在宿主的抗肿瘤作用中起作用。 天生的反应据报道MERS辅助蛋白NS 4a和NS 4 b拮抗干扰素（IFN）-β 在过表达和报道系统中诱导，我们在此提供的数据显示了 蛋白质赋予重组突变MERS病毒复制的衰减。然而，人们对 MERS感染期间宿主拮抗作用的机制，这是另一个重要的知识空白。NS4a 是一种dsRNA结合蛋白，定位于病毒复制/转录复合物并拮抗IFN-λ NS 4 b与NCBI中的其它蛋白均无同源性。我们用结构模型 将MERS NS 4 b鉴定为LigT样2 H-磷酸酯酶（2 H-PE），并且类似于谱系A的NS 2蛋白 β冠状病毒MHV，NS 4 b具有2 '，5'-磷酸二酯酶（PDE）活性，并拮抗RNA酶L， 细胞质然而，与NS 2不同，MERS NS 4 b具有N-末端核定位信号（NLS）， 主要集中在细胞核。在初步数据中，NS 4 b还切割在可能的RNA中发现的3 '，5'键。 基质，暗示其他可能的核功能。RNA-seq数据表明NS 4 b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 选择mRNA的修饰。我们将检验MERS NS 4a和NS 4 b拮抗dsRNA的假设。 NS 4 b是一种独特的冠状病毒蛋白， 在细胞核中酶促表达以下调选择的宿主mRNA的丰度， 对抗抗病毒反应。我们建议：1.使用重组MERS突变病毒评估NS 4a 和NS 4 b介导的拮抗dsRNA诱导的抗病毒途径在人A549细胞和原代 人气道上皮细胞2.研究NS 4 b的底物特异性以及其预测的核转录因子。 在转录后调节选择的抗病毒mRNA丰度的作用，并探讨其可能性 NS 4 b调节程序性细胞死亡。3.通过感染蝙蝠鉴定蝙蝠特异性MERS-宿主相互作用 用MERS和NS 4a和NS 4 b突变体病毒在体内对衍生的细胞系和蝙蝠进行了研究。这些研究将阐明 MERS NS 4a和NS 4 b辅助蛋白的可能的多种功能，并在长期导致 候选治疗靶点的鉴定。此外，这些发现可能有助于解释 人畜共患病病毒感染人类的致病结果与其自然宿主相比。

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.