Control of coronavirus pathogenesis by antagonism of RNase L

 描述（由申请人提供）：人类冠状病毒（HCoV）-OC 43和HCoV-229 E长期以来一直被认为是普通感冒的病原体。2003年SARS-CoV作为严重急性呼吸综合征的病原体出现，最近中东呼吸综合征（MERS）-CoV的死亡率分别为10%和30%，这使得了解冠状病毒与宿主的相互作用对毒力的贡献变得至关重要。病毒逃避或拮抗I型干扰素（IFN）应答的能力在发病机制中起着至关重要的作用。冠状病毒免疫逃避的一个未充分研究的方面是寡腺苷酸合成酶（OAS）-核糖核酸酶（RNase）L途径的拮抗作用，这是一种IFN诱导的有效抗病毒机制。一旦被双链RNA激活，OAS合成2 '，5'-连接的寡腺苷酸（2-5A），其激活RNA酶L。RNA酶L切割单链RNA，导致病毒基因组和宿主RNA的降解、蛋白质合成停滞和细胞凋亡。2a组β冠状病毒，小鼠肝炎病毒（MHV）辅助蛋白ns 2是一种2 '，5'-磷酸二酯酶（PDE），其切割2-5A，从而防止RNA酶L的活化。PDE活性是Kupffer细胞和肝窦内皮细胞复制以及小鼠肝毒性的关键决定因素。其他2a组β冠状病毒，包括HCoV-OC 43，编码ns 2同源物，我们最近证实为RNA酶L拮抗剂。此外，2c组β冠状病毒MERS-CoV辅助蛋白NS 4 b被预测为PDE，并且在初步实验中可以降解2-5A。这些发现导致以下假设：2c组和2a组β冠状病毒通过PDE介导的2-5A切割拮抗OAS-RNase L途径，并且OAS-RNase L拮抗作用有助于人β冠状病毒在呼吸道中的复制，并可能导致发病机制。目的1将研究MERS-CoV NS 4 b和相关组2c Bat-CoV同源物的预测PDE活性。重组NS 4 b和同源物的PDE活性将通过2-5A切割测定在体外评估，并通过测量核糖体RNA完整性和定量2-5A水平在转染细胞中评估。此外，将构建表达无活性ns 2和每种活性NS 4 b PDE的嵌合MHV，并评估复制和肝炎的拯救。最后，将评估核定位的影响，信号位于NS 4 b上，而不存在于2a组β冠状病毒PDE上。目的2将检查RNase L拮抗作用对HCoV-OC 43和MERS-CoV在原代人气道细胞（包括上皮细胞、内皮细胞和巨噬细胞）中成功复制的贡献。用于分析拮抗作用的方法将包括病毒滴度和核糖体RNA完整性。结果将与这些细胞类型中OAS基因的基础表达水平相关。这项研究将有助于了解冠状病毒-宿主相互作用，特别是PDE介导的RNase L拮抗作用对人类呼吸道冠状病毒感染的影响。最后，病毒编码的PDE可能为抗病毒治疗提供诱人的靶点。

 DESCRIPTION (provided by applicant): Human coronaviruses (HCoV)-OC43 and HCoV-229E have long been known as agents of the common cold. The emergence of SARS-CoV as the etiological agent of severe acute respiratory syndrome in 2003 and more recently, Middle East respiratory syndrome (MERS)-CoV with 10% and 30% mortality respectively, make it imperative to understand the coronavirus-host interactions that contribute to virulence. The ability of viruses to evade or antagonize the type I interferon (IFN) response plays a vital role i pathogenesis. An understudied aspect of immune evasion by coronaviruses is the antagonism of the oligoadenylate synthetase (OAS)- ribonuclease (RNase) L pathway, an IFN-induced potent antiviral mechanism. Once activated by double- stranded RNA, OAS synthesizes 2',5'-linked oligoadenylates (2-5A) that activate RNase L. RNase L cleaves single-stranded RNA leading to degradation of viral genomes and host RNA, protein synthesis arrest, and apoptosis. The group 2a Betacoronavirus, mouse hepatitis virus (MHV) accessory protein ns2 is a 2',5'- phosphodiesterase (PDE), which cleaves 2-5A thereby preventing activation of RNase L. PDE activity is a critical determinant of replication in Kupffer cells and liver sinusoidal endothelialcells as well as hepatovirulence in mice. Other group 2a Betacoronaviruses, including HCoV-OC43, encode ns2 homologs that we have recently confirmed as RNase L antagonists. Additionally, the group 2c Betacoronavirus MERS-CoV accessory protein NS4b is predicted to be a PDE and in preliminary experiments can degrade 2-5A. These findings lead to the hypotheses that group 2c as well as group 2a Betacoronaviruses antagonize the OAS-RNase L pathway by PDE-mediated cleavage of 2-5A and that OAS-RNase L antagonism contributes to human Betacoronavirus replication in the respiratory tract, and potentially to pathogenesis. Aim 1 will investigate the predicted PDE activity of MERS-CoV NS4b and related group 2c Bat- CoV homologs. PDE activity of recombinant NS4b and homologs will be assessed in vitro by a 2-5A cleavage assay and in transfected cells by measuring ribosomal RNA integrity and quantifying 2-5A levels. In addition, chimeric MHVs expressing inactive ns2 and each active NS4b PDE will be constructed and the rescue of replication and hepatitis assessed. Finally, the impact of the nuclear localization, signal located on NS4b and not present on the group 2a Betacoronavirus PDEs, will be assessed. Aim 2 will examine the contribution of RNase L antagonism to the success of HCoV-OC43 and MERS-CoV replication in primary human airway cells including epithelial, endothelial and macrophages. Metrics for analysis of antagonism will include viral titers and ribosomal RNA integrity. Results will be correlated with basal expression levels of OAS genes in these cell types. This study will contribute to the understanding of coronavirus-host interactions, in particular the impact of PDE-mediated RNase L antagonism on human respiratory coronavirus infection. Finally, virus-encoded PDEs potentially provide a tantalizing target for antiviral therapeutics.