N~6-methyladenosine Modulates Enterovirus 71 Replication

N~6-methyladenosine(m~6A)is the first reversible RNA modification discovered.It constitutes the most prevalent modification in RNA and is critical in RNA metabolism and function.It has been found that viral RNA contains m~6A modifications as early as40 years ago,but due to technical and intellectual limitations,the function of viral m~6A is not clear.Until recently,it has been reported that HIV,Influenza virus and Flavivirus contain m~6A nucleotides which modulate viral gene expression and replication.However,there are few studies on the function of viral m~6A modification and the molecular mechanism is unclear.The research of m~6A will remain a hot and difficult topic in epigenetics of viruses in the future.To study the function of m~6A modification in EV71 RNA,we first used UHPLC,MeRIP-Northern Blot and MeRIP-Seq to prove that the protein coding regions of VP,2C and 3D on EV71 RNA contain m~6A modifications.Subsequently,we found that EV71 infection increases the expression of m~6A methyltransferase and binding proteins but decreases the expression of demethylase by using Western Blot.At the same time,immunofluorescence results showed that the cellular localization of these proteins has changed after EV71 infection.Next,in order to test the effect of m~6A modification on virus replication,we knocked down m~6A methyltransferase and demethylase,which resulted in decreased and increased EV71 replication,respectively.In addition,the replication of EV71 was also significantly affected when m~6A binding proteins were knocked down.To further verify the function of m~6A,we mutated the m~6A sites on EV71 RNA and the results showed that viral replication was significantly inhibited.These results indicated that m~6A modification affects viral replication.Later,we tried to explore the molecular mechanism of the impact of m~6A modification system on virus.Mass spectrometry results showed that methyltransferase METTL3 interacts with the EV71 3D protein,and Western Blot and immunofluorescence further confirmed this interaction.We also found that the stability of 3D protein is affected by changing the expression of METTL3.Since it has been reported that SUMOylation and ubiquitination affect the stability of 3D protein and RNA polymerase activity,METTL3 may modulate viral replication through this pathway.Sumoylation and ubiquitination assays revealed that METTL3promotes 3D SUMOylation and ubiquitination,suggesting that METTL3 can regulate3D protein modification,the status of which affects viral replication.In summary,we found that EV71 RNA contains m~6A modification,and the m~6A modification system plays an important role in viral infection.Our research is conducive to further understand the function of viral m~6A modification,and can be used for reference for the study of viral epigenetics in the future.In addition,our study provides new ideas and possible drug targets for virus prevention and treatment.