Cell-Based Assays To Detect Inhibitors Of Coronavirus Methyltransferase And Construction Of Coronavirus Reverse Genetics System

Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7(cap0) and2’-O (cap1) positions of the5’guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. The cap has several important biological roles, such as protecting mRNA from degradation by5’exoribonucleases, directing pre-mRNA splicing and mRNA export from the nucleus and conferring stability to mRNAs and ensuring their efficient recognition for translation. In addition,2’-O-methylguanosine (m7GpppmA) cap is the structural feature of Coronavirus mRNA and it is required for evading the recognization by RIG-I like Virus dsRNA specific receptor MDA5(Pattern Recognition Receptors) and the immune respose mediated by interferon inducible protein(IFIT). The capping mechanisms of some viral mRNAs are different from those seen for the cellular mRNA. The differences between host and viral cap formation could potentially be used for development of antiviral therapy. Howere, it is necessary to build an system to screen inhibitors for coronavirus methylation and make it clear if or not N7-methylation of coronavirus mRNA is avaliable for protecting viral mRNA from recognization by cellular PRRs.In this article, we describe the development of yeast cell-based assays to identify Coronavirus inhibitors of mRNA capping using the yeast YBS40strain developed by Shuman and colleagues. The YBS40strain was transformed Coronavirus mRNA methyltransferase to successfully growth under5-FOA negative selection. We use this system to screen different chemical collections in96or384microplate by Spectrophotometry monitoring the yeast growth. Our system makes screening of Coronavirus mRNA capping inhibitor rapidly and sensitively and could be readily adapted for high-throughput. There are4out of5,000natural products which was extracted in2,000actinomyces (WS) and fungi (SF) could be inhibited the activity of coronavirus or fungi N7-MTase using our screening system.We develop the Reverse genetics of Murine Hepatitis Virus (MHV-A59) using vaccinia virus as a vector, in which critical residues in N7-MTase and3’-5’exonuclease is mutated and lose its activity. Then we attempt to introduct those point mutations into MHV-A59nsp14and got four MHV-nsp14-D89&E91A, D330A, C408R and Y414A mutants. In previous research, MHV-D89A&E91A mutant strains lose their exonuclease activity but the methylation not affected; MHV-D330loses N7methyltransferase activity and affects the replication of SARS replicon; MHV-C408R is an ts mutant and its N7-MTase activity run well at30℃but not37℃; MHV-Y414A attenuated virulence and reduced titers in vivo and loses N7-MTase activity. However, it is interesting that we found the plaque size of MHV-D89A&E91A was smaller than MHV-A59wild type and there is no plaque of MHV-Y414A at39.5℃showing it’s a ts mutant strain.To sum up, we develop a good system for screening the inhibitors of Coronavirus mRNA capping and it performs well in96well and384-well plate. Furthermore, the system possesses several advantages compared with other assays, including a simple experimental protocol, low sample consumption and the ability to use the traditional Spectrophotometry without the need for optimizations of specific reaction conditions. The increasing knowledge about active-site differences between cellular and viral MTases is expected to provide antiviral selectivity. And in addition, there are four MHV-A59mutants to ued to study the fidelity of coronavirus genomes and evading the recoganition by the host innate immunity.