Study On The Role And Mechanism Of M6A Methylation Modification In JEV Infection

Japanese encephalitis virus(JEV)is a single plus-stranded RNA virus of flavivirus genus of flaviviridae.Japanese encephalitis is a serious nervous system disease caused by JEV infection with high mortality and disability rate.Although there is vaccination for prevention,due to the increased mobility of the population and the impact of the virus itself variation,vaccine protection is limited,and there is still a lack of specific treatment drugs in clinical practice.The pathogenesis of JEV has not been fully elucidated,which is the bottleneck of clinical drug development at present.N6-methyladenosine(m6A)is a reversible and most widely distributed RNA modification,which plays an important role in RNA metabolism and a variety of biological processes.A number of studies have confirmed that m6 A modification plays an important regulatory role in the pathogenesis of a variety of virus infections,but its regulatory mechanism on JEV infection remains to be cleared.Therefore,this study took JEV as the research object to preliminatively explore the regulatory role of m6 A methylation modification in JEV infection and pathogenicity,and the details are as follows.First,methylated RNA immunoprecipitation sequencing revealed that the protein coding regions of JEV genome RNA,such as E and NS5,contained m6 A modifications.M6 A modification in the host is mainly concentrated in the CDS region and the 3’UTR front end of the encoding gene.The dominant motif in host RNA was "CGAC".Secondly,Neuro2 a,N9 and BHK cells were infected with JEV,and the expression of m6 A modified molecules was detected by Western Blot.The results showed that the expression of writer METTL3 and reader YTHDF1 was first increased(24h after JEV infection)and then decreased(48h after JEV infection).Then JEV was injected into the brain of Suckling mice,and the expression of M6A-related molecules in brain tissues was detected by Western Blot.The results also showed that METTL3 and YTHDF1 increased first and then decreased.To further analyze the effect of m6 A modification on JEV replication,we found that JEV replication was significantly inhibited on Neuro2 a cells by si RNA knockdown of METTL3 and YTHDF1.When METTL3 and YTHDF1 were overexpressed,the replication of JEV was promoted.In addition,we treated JEV-infected cells and mice with the m6 A methylation inhibitor 3-DAA.It was found that 3-DAA treatment promoted JEV replication at the cellular level.At the animal level,3-DAA significantly accelerated the pathogenesis and increased the mortality of JEV-infected mice.Further studies showed that 3-DAA treatment could downregulate the expression of il-6,TNF-α,CXCL10 and other inflammatory factors.Finally,in order to preliminarily explore the possible mechanism of m6 A modification and regulation of JEV replication,we infected METTL3 and YTHDF1 Neuro2 a cells with JEV respectively and knocked them down.The expression of interferon-related molecules was detected by QRT-PCR.The results showed that IFN-β,IRF7,Ifit1,ISG15 and other interferon-related molecules were increased,suggesting that m6 A modification may regulate the replication of JEV through interferon signaling pathway.In conclusion,this study confirmed that m6 A modification exists in JEV genome,and JEV infection affects the expression of m6A-related molecules.m6 A modification can positively regulate the replication of JEV,and its regulatory mechanism may play a role through interferon pathway.Our study preliminarily elucidated the pathogenesis of JEV from the perspective of epigenetics,providing new ideas and potential therapeutic targets for the prevention and treatment of the virus.