Study On The Relationship Between The Stability Of Mouse Hepatitis Virus S Protein And Virus Adaptability And Its Expression Mechanism

Coronaviruses(CoVs)are a family of enveloped and single positive-stranded RNA viruses with the largest known RNA genomes,approximately 30 Kb.CoVs infect varieties of species including human,avian,and mammals causing diseases in upper respiratory tract,gastro-intestinal tract,liver and central neural system.The outbreaks of Severe Acute Respiratory Syndrome(SARS)in China in 2003 and Middle East Respiratory Syndrome(MERS)in the Middle East in 2012 have raised the importance and urgency of studying CoVs.CoV utilizes the viral spike(S)protein for virus entry into host cell.My thesis aims to elucidate the roles of S protein of mouse hepatitis virus A59 strain(MHV-A59)in following two aspects:1)The relationship between S protein stability and virus fitness;2)How virus evades the surveillance of host RNA decay pathways,especially nonsense-mediated mRNA decay,to express S protein.Enveloped virus gains entry into host cell by fusion of viral and cellular membranes,which is mediated by the interaction between viral envelope proteins and host receptors.In general,S protein of CoV is metastable prior to binding to its receptor,and activation of conformational changes of S protein is under the strict control because improper triggering at the wrong timing and location will result in the loss of infectivity.In this study,we found that histidine at the position 209 of S protein of MHV A59 greatly influences the thermal stability of S protein.A single H209A mutation significantly increased the thermal stability of S protein,but it markedly decreased virus fitness,indicating that the higher stability of S protein may not result in greater fitness for virus.This study explained the reason why His209 was kept in MHV A59 during the evolution.In eukaryotic cells,mRNAs are under strict quality control,Several RNA decay pathways consistently monitor and degrade the abnormal mRNAs.Non-sense mediated RNA decay is one of host RNA decay pathway,and it mainly targets mRNAs with premature translation termination codons(PTCs).However,recent studies showed that mRNA with multiple open reading frames(ORFs)or very long 3' untranslated region(3'UTR)also could be targeted by NMD.CoV genome and most of its subgenomes contains multiple ORFs in a single RNA and also have long 3'UTR,which are characteristics of NMD substrates.In this study,we found that knocking-down either UPF1,UPF3,or SMG7,the essential components of NMD,significantly enhanced viral production,suggesting that CoV RNAs are targeted by NMD.However,host NMD activity was significantly inhibited by overexpression of either viral N protein or host PTBP1.Moreover,viral N protein physically interacted with PTBP1,knocking-down PTBP1 by siRNA significantly decreased virus production.Finally,addition of either MHV 5' or 3' UTRs to a NMD sensitive substrate markedly suppressed RNA degradation by NMD,indicating that there are NMD-inhibitory cis elements present in MHV 5' and 3' UTRs.