Characterization Of The Guanine-N7 Methyltransferase Activity Of Porcine Epidemic Diarrhea Virus And M~6A Modification Of Delta Coronavirus

Porcine epidemic diarrhea virus(PEDV)is a member of a Coronavirus,and Porcine Delta Coronavirus belongs to Delta Coronavirus,which could cause vomiting,diarrhea and dehydration in pigs,especially newborn piglets within 1 week.The morbidity and mortality of newborn piglets infected could reach up to 100%.These viruses could cause severe economic loss to swine-breeding industry.Coronavirus is single-strand positive RNA virus that carries a 5' capping structure which has important effects on RNA stability,nuclear export,translation of RNA,espically evasion of detection by natural immune system.And RNA interior modification--m6A also plays an important role in regulation of transcription and stability of RNA.RNA capping structure and m6A modification could be the target for antiviral medicines or vaccines.However,there are few correlated researches on a and ?coronaviruses about methylated modification,so the role of PEDV G-N7 MTase and PDCoV m6A modification in virus replication is intended to study.For coronaviruses,RNA capping structure is firstly methylated at guanine N-7(G-N7)position by nonstructural protein 14(nsp14),which facilitates and precedes the subsequent ribose 2'-O methylation by nspl6-nsp10 complex.Using porcine epidemic diarrhea virus(PEDV),an Alphacoronavirus,as a model,we showed that G-N7 methyltransferase(MTase)of PEDV nsp14 methylates RNA substrate in a sequence-unspecific manner.PEDV nsp14 could efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environment,and can methylate cap analogs(GpppA and GpppG)and single nucleotide GTP but not ATP,CTP,or UTP.Site-directed mutations in PEDV nsp14 were used to study the crucial amino acid for G-N7 MTase.Mutations to the Sadenosyl-L-methionine(SAM)binding motif(aa330-aa335)in the nsp14 almost abolished the G-N-7 MTase activity and were lethal to PEDV.However,recombinant nsp14 protein with a single mutation(D350A)in nsp14 retained 29.1%of G-N-7 MTase activity.These results showed that crucial amino acid residues in the S-adenosyl-L-methionine(SAM)binding motif in the nsp14 were necessary for G-N7 MTase.Recombineering system was used to construct mutant PEDV cDNA clone through Two-step selection/counter-selection method.The first positive selection step is relatively easy to perform,the majority(80%)of Kanamycin-resistant colonies contained recombineering products.However,the second counter-selection step for streptomycin sensibility is relatively hard to perform,only 0.5%-3%of colonies contained required recombineering products.Mutations to the S-adenosyl-L-methionine(SAM)binding motif(aa330-aa335)in the nsp14 almost abolished the G-N7 MTase activity and were lethal to PEDV.However,recombinant rPEDV-D350A with a single mutation(D350A)in nsp14,which retained 29.1%of G-N-7 MTase,was viable.Compared with parental rPEDV,recombinant rPEDV-D350A formed significant smaller plaques and had significant defects in viral protein synthesis and viral replication in Vero-CC181 cells and intestinal porcine epithelial cells(IPEC-DQ)cells.Notably,rPEDV-D350A could induced significantly higher expression of type ? and ? interferons in IPEC-DQ cells compared to the parental rPEDV.Collectively,our results demonstrate that G-N7 MTase of PEDV modulates viral replication,gene expression,and innate immune responses.Besides RNA capping structure,m6A modification has important effect on stability and translation efficiency of RNA.There are many reports about m6A functions in host cells not Coronaviruses.The relationship between m6A and virus infection was studied in this experiment by Using porcine Delta Coronavirus(PDCoV)as a model.Thees results showed that m6A writers,erasers and readers could affect the replication of PDCoV.The infection of PDCoV has no effect on the quantity of m6A writers,erasers and readers,but it could promote spliceosomes production.YTHDF3 re-located to RTC where virus replicates and translates,and it is co-located with N protein and virus RNA.The data of RIP showed YTHDF3 could recognize and bind to genome RNA of PDCoV.These data show that m6A modification has positive effect on replication of PDCoV,and YTHDF3 plays potential roles on stabilizing viral RNA,promoting viral RNA replication and translation,and ultimately facilitating viral assembly,but more detailed mechanisms remain to be elucidated and discussed by further studies.In this study,PEDV cDNA infectious clone based on Red recombineering system could be useful in further study of viral genome.PEDV nsp14 G-N7 MTase plays crucial role in replication and transcription of virus,and G-N7 modification could regulate immune response.m6A modification also could regulate viral replication and protein expression.RNA capping structure and m6A modification could be the target for antiviral medicines or vaccines.