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

研究 MERS-CoV NS4b 作为细胞核内宿主抗病毒反应的调节剂

基本信息

批准号：
9619001
负责人：
Stephen Goldstein
金额：
$ 2.36万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2019-05-20
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9619001
关键词：
A549 Amino Acids Antiviral Agents Antiviral Response Biological Biological Assay Biology CRISPR/Cas technology Cell Death Cell Line Cell Nucleus Cells Cessation of life Cleaved cell Co-Immunoprecipitations Conflict (Psychology)Confocal Microscopy Coronavirus Cytoplasm Data Dipeptidyl-Peptidase IV Distributional Activity Ectopic Expression Engineering Ensure Enzyme-Linked Immunosorbent Assay Enzymes Epithelial Exhibits Exons Family Gene Expression Genetic Transcription Genome Histidine Homologous Gene Human IRF3 gene Immune response Importins Infection Interferon-beta Interferons Introns Kinetics Knock-out Label Link Literature Liver Mediating Mediator of activation protein Messenger RNA Middle East Respiratory Syndrome Coronavirus Molecular Biology Techniques Murine hepatitis virus Mus Mutate Myeloid Cells Nuclear Nuclear Import Nuclear Translocation Pathogenesis Pathway interactions Physiological Play Process Proteins Public Health RNA Recombinants Reporter Reporting Research Research Personnel Ribonucleases Role SARS coronavirus Sequence Homology Severe Acute Respiratory Syndrome Stains Structural Models Testing Time Training Transcript Transcription Process Viral Viral Proteins Virulent Virus Virus Replication Western Blotting Work Zoonoses arm base career experimental study heterokaryon leptomycin B mutant novel pathogen phosphodiester phosphodiesterase V promoter receptor reverse genetics success therapeutic target transcriptome sequencing transmission process

项目摘要

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.

项目摘要中东呼吸综合征冠状病毒（MERS-COV/MERS）是最近出现的人畜共患病原体自2012年发现以来，这造成了近1,800例病例和600多人死亡。 MERS是第二个在2003年SARS-COV（SARS）之后，在过去的十五年中出现了强烈的冠状病毒证明冠状病毒是对全球公共卫生的持续威胁。而Mers，Sars和鼠冠状病毒均属于同一属，贝塔科罗纳病毒，MERS与两者都有很高的不同它的基因组编码了一组新型的辅助蛋白。所有病毒必须操纵宿主的潜水员武器抗病毒反应以确保复制成功和传播，以及冠状病毒附件蛋白参与这些基本功能。由于它的新颖性和与先前研究的差异冠状病毒，MERS辅助蛋白在很大程度上没有表征，并且缺乏明显的序列与其他已知病毒蛋白的同源性。我们已经使用结构建模来识别MERS NS4B 辅助蛋白作为小鼠肝炎病毒（MHV）NS2辅助蛋白的同源物，先前由我们的实验室描述为抗病毒酶RNase L的拮抗剂。这两种蛋白都是同源物在LIGT样的2H磷酸酯酶（2H-PES）中，这是一个大型原核和真核蛋白的家族由2 HXS/T催化基序被80-100氨基酸隔开。 NS2对于MHV复制至关重要在髓样细胞和小鼠肝脏中，NS4B酶促活性的失活导致RNase L激活在人类气道上皮3细胞的MERS感染期间。 MHV NS2或 MERS NS4B取决于由催化组氨酸介导的2'，5'磷酸二酯酶活性。尽管有这两种蛋白质之间的相似性，我们的实验室已经确定了引人注目的差异和显着的初步数据与文献中的报道结合了NS4B可以拮抗干扰素β的表达记者测定中的启动子表明，与MHV NS2相比，它具有额外的功能。值得注意的是 MHV NS2仅是细胞质的，MERS NS4B包含一个强大的功能性NLS，将主要定位为核us。我们的实验室以前证明，核定位是完全可分配的拮抗RNase L，NS4B NLS在所有已知的MERS样病毒中配置，强烈支持核功能的想法。另外，而MHV NS2仅切割2'-5'连接的磷酸二酯键，但 MERS NS4B裂解2'-5'和3'-5'链接债券，使其具有更广泛的基板范围和能力众所周知，某些原核和真核2H-PE的过程RNA。我们的初步数据强烈建议NS4B转录后处理与抗病毒反应和细胞相关的mRNA 死亡，通过其酶促活性在核中这样做。因此，MERS NS4B可能是第一个已知的病毒2H-PE在细胞核中起作用。这项研究可能会阐明冠状病毒的新机制与宿主相互作用，并确定NS4B在MERS感染过程中是否是合理的治疗靶标。