Control of coronavirus pathogenesis by antagonism of RNase L

 描述(申请人提供)：人类冠状病毒(HCoV)-OC43和HCoV-229E长期以来一直被认为是普通感冒的病原体。2003年SARS冠状病毒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)冠状病毒的死亡率分别为10%和30%，这使得了解冠状病毒与宿主之间的相互作用成为当务之急。病毒逃避或拮抗I型干扰素反应的能力在I型干扰素的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被研究的方面是拮抗寡核苷酸合成酶-核糖核酸酶L途径，这是干扰素诱导的一种有效的抗病毒机制。OAS一旦被双链核糖核酸激活，就会合成2‘，5’-连接的寡腺苷(2-5A)，激活核糖核酸酶L-核糖核酸酶L裂解单链核糖核酸，导致病毒基因组和宿主核糖核酸的降解，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NS2是一种2‘，5’-磷酸二酯酶(PDE)，它能裂解2-5A从而阻止核糖核酸酶L的激活。PDE活性是Kupffer细胞和肝窦内皮细胞复制以及小鼠肝脏毒力的关键决定因素。其他2a组Betacorona病毒，包括人类冠状病毒OC43，编码我们最近确认为核糖核酸酶L拮抗剂的NS2同源物。此外，2c组BetacoronaVirus MERS-CoV辅助蛋白NS4B被预测为PDE，在初步实验中可以降解2-5A。这些发现导致假设2c组和2a组β-冠状病毒通过PDE介导的2-5A的裂解来拮抗OAS-核糖核酸酶L途径，并且OAS-核糖核酸酶L拮抗作用参与了人β冠状病毒在呼吸道的复制，并可能参与了病毒的致病。目的1研究MERS冠状病毒NS4b及其相关的2c BAT冠状病毒同源物的PDE活性。重组NS4B及其同源物的PDE活性将通过2-5A切割试验在体外进行评估，并通过测量核糖体RNA完整性和定量2-5A水平在转基因细胞中进行评估。此外，还将构建表达非活性NS2和每个活性NS4B PDE的嵌合MHV，并评估对复制和肝炎的抢救。最后，将评估核定位的影响，该信号位于NS4B上，不存在于2a组BetacoronaVirus PDE上。目的2研究核糖核酸酶L拮抗剂在人呼吸道上皮细胞、内皮细胞和巨噬细胞中复制HCoVOC43和MERS冠状病毒的作用。分析拮抗作用的指标将包括病毒滴度和核糖体RNA完整性。结果将与OAS基因在这些细胞类型中的基础表达水平相关。本研究将有助于了解冠状病毒与宿主的相互作用，特别是PDE介导的核糖核酸酶L拮抗剂对人呼吸道冠状病毒感染的影响。最后，病毒编码的PDE可能成为抗病毒治疗的诱人靶点。

 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.