Investigating MERS-CoV NS4b as a modulator of the host antiviral response in the nucleus

项目摘要 中东呼吸综合征冠状病毒（MERS-CoV/MERS）是一种新近发现的人畜共患病原体 自2012年发现以来，已造成近1,800例病例和600多人死亡。MERS是第二个 继2003年SARS-CoV（SARS）之后，过去十五年中出现了致命的冠状病毒，进一步 这表明冠状病毒是对全球公共卫生的持续威胁。而MERS、SARS和 鼠冠状病毒都属于同一属，β冠状病毒，MERS与两者高度不同， 其基因组编码一组新的辅助蛋白。所有病毒都必须操纵宿主的不同分支 确保复制成功和传播的抗病毒反应，以及冠状病毒辅助蛋白 参与这些重要的功能。由于其新奇和与先前研究的分歧， MERS辅助蛋白在很大程度上是未表征的，并且缺乏重要的序列 与其他已知病毒蛋白同源。我们已经使用结构建模来识别MERS NS 4 b 辅助蛋白作为小鼠肝炎病毒（MHV）NS 2辅助蛋白的同源物， 以前被我们的实验室描述为抗病毒酶RNase L的拮抗剂。两种蛋白质都是同源物 LigT-like 2 H-phosphoesterases（2 H-PEs）是原核和真核生物蛋白质的一个大家族 其特征在于由80-100个氨基酸分开的2个HxS/T催化基序。NS 2对MHV复制至关重要 在骨髓细胞和小鼠肝脏中，NS 4 b酶活性的失活导致RNase L活化 在MERS感染人气道上皮Calu-3细胞期间。MHV NS 2或MHV NS 3对RNase L的拮抗作用 MERS NS 4 b依赖于由催化组氨酸介导的2 '，5'磷酸二酯酶活性。尽管 这两种蛋白质之间的相似性，我们的实验室已经确定了显着的差异和重要的初步数据 结合文献中NS 4 b可以拮抗干扰素- β的表达， 启动子在报告基因测定中的表达表明，与MHV NS 2相比，它具有额外的功能。值得注意的是， MHV NS 2仅在细胞质中，MERS NS 4 b含有强的功能性NLS，并主要定位于 原子核我们的实验室以前证明，核定位是完全不可能的。 NS 4 b NLS在所有已知的MERS样病毒中是保守的，强烈支持了 核函数的想法。此外，尽管MHV NS 2仅切割2 '-5'连接的磷酸二酯键， MERS NS 4 b裂解2 '-5'和3 '-5'连接键，使其具有更广泛的底物范围和能力， 一些原核和真核的2 H-PE也是这样。我们的初步数据显示 表明NS 4 b转录后加工与抗病毒反应和细胞 死亡，通过其酶活性在细胞核中这样做。因此，MERS NS 4 b可能是第一个已知的 病毒2 H-PE作用于细胞核。这项研究可能阐明了冠状病毒 与宿主相互作用，并确定NS 4 b是否是MERS感染期间的合理治疗靶点。

Project Summary Middle East Respiratory Syndrome coronavirus (MERS-CoV/MERS) is a recently emerged zoonotic pathogen that has caused almost 1,800 cases and over 600 deaths since its 2012 discovery. MERS is the second virulent coronavirus to emerge in the last fifteen years, following SARS-CoV (SARS) in 2003, further demonstrating that coronaviruses are a continuing threat to global public health. While MERS, SARS, and the murine coronaviruses all belong to the same genus, Betacoronavirus, MERS is highly divergent from both and its genome encodes a novel set of accessory proteins. All viruses must manipulate diverse arms of the host antiviral response to ensure replicative success and transmission, and coronavirus accessory proteins participate in these essential functions. Due to its novelty and divergence from previously studied coronaviruses, the MERS accessory proteins are largely uncharacterized and lack significant sequence homology with other known viral proteins. We have used structural modeling to identify the MERS NS4b accessory protein as a homolog of the mouse hepatitis virus (MHV) NS2 accessory protein, which was previously described by our lab as an antagonist of the antiviral enzyme RNase L. Both proteins are homologs of the LigT-like 2H-phosphoesterases (2H-PEs), a large family of prokaryotic and eukaryotic proteins characterized by 2 HxS/T catalytic motifs separated by 80-100 amino acids. NS2 is critical for MHV replication in myeloid cells and the mouse liver, and inactivation of NS4b enzymatic activity results in RNase L activation during MERS infection of human airway epithelial Calu-3 cells. Antagonism of RNase L by either MHV NS2 or MERS NS4b is dependent on 2’,5’ phosphodiesterase activity mediated by the catalytic histidines. Despite the similarity between these two proteins, our lab has identified striking differences and significant preliminary data which, combined with reports in the literature that NS4b can antagonize expression from the interferon- β promoter in reporter assays, suggest it has an additional function as compared to MHV NS2. Notably, whereas MHV NS2 is exclusively cytoplasmic, MERS NS4b contains a strong, functional NLS and localizes primarily to the nucleus. Our lab previously demonstrated that nuclear localization is completely dispensable for antagonizing RNase L, and the NS4b NLS is conserved in all known MERS-like viruses, strongly supporting the idea of a nuclear function. Additionally, whereas MHV NS2 cleaves only 2’-5’ linked phosphodiester bonds, MERS NS4b cleaves both 2’-5’ and 3’-5’ linked bonds, giving it broader substrate range and the ability to process RNA, as some prokaryotic and eukaryotic 2H-PEs are known to do. Our preliminary data strongly suggest that NS4b post-transcriptionally processes host mRNAs associated with the antiviral response and cell death, doing so in the nucleus through its enzymatic activity. Therefore, MERS NS4b is likely the first known viral 2H-PE to act in the nucleus. This research may illuminate a new mechanism by which coronaviruses interact with the host, and determine whether NS4b is a plausible therapeutic target during MERS infection.