Effects Of CVA16 Infection On M~6A Methylation-related Protein Expression And Localization

Coxsackievirus A 16(CVA16)and Enterovirus 71(EVA71)in enterovirus A are currently the most important pathogens that cause hand-foot-mouth disease in the whole world,all of which belong to positive-strand RNA viruses,with highly homologous in nucleotide sequence and amino acid sequence.In recent years,CVA16 and EVA71 have undergone gene recombination and co-prevalence,which aggravated the incidence and clinical symptoms of HFMD.The inactivated EVA71 vaccines that have already been marketed cannot play a cross-protective role on CVA16.N6-methyladenylate(m6A)is a methylated modification of the 6th N atom of adenine.It is mainly distributed on the 3'non-coding region,translation start site and stop codon on the mRNA,which can be controlled RNA splicing,translation and stability play a key role in RNA metabolism and function.At present,there have been studies that found m6A modification in the viral genome of EVA71,and also proved that m6A methylation-related proteins can regulate EVA71 replication.However,no studies have been reported on m6A modification after CVA16 infection.In this study,we first selected human respiratory epithelial cells(16HBE cells)that enteroviruses first invaded through the respiratory route,and analyzed the proliferation kinetics of CVA16 and EVA71 on 16HBE cells.Then explore whether the virus infection of 16HBE cells,ICR suckling mice and mice expressing human SCARB2 receptor will affect the expression of methyltransferase,demethylase and methylated reading protein in m6A modification,and Localization in 16HBE cells under normal conditions and after virus infection.CVA16 and EVA71 infected 16HBE cells with MOI=0.1,and conducted the following experiments respectively:(1)Collect samples at 12h,24h and 48h after infection for infectious titer determination(3 repeated trials);(2)Using non-infected cells as a 0 hour sample,Western Blot was used to detect the expression of viral structural protein VP1 after CVA16 and EVA71 infection;(3)Using immunofluorescence technology to detect the infection rate of CVA16 and EVA71 on 16HBE cells;(4)Immunofluorescence and fluorescence confocal microscopy were used to observe the co-localization of m6A methyltransferase,demethylase and methylated reading protein with VP1 after CVA16 and EVA71 infection.PBS,CVA16 and EVA71 respectively were injected into the cranial cavity of 1-day-old ICR suckling mice,and the PBS group was used as the control group to perform the following experiments:(1)Observe the daily incidence of suckling mice,record the number of dead suckling rats per day,and draw the survival curve of each group;(2)Western Blot was used to detect the expression of methyltransferase,demethylase and methylated reading protein in the brain tissue of ICR suckling mice.PBS and CVA16 respectively were infected by nasal infection with hSCARB2-transgenic mice,and the PBS group served as the control group Western Blot was used to detect the expression of m6A methylation-related protein in the brain tissue of hSCARB2-transgenic mice.The study found that,(1)both CVA16 and EVA71 can replicate and proliferate on 16HBE cells;(2)With the increase of viral infection time,the expression level of m6A methylation-related proteins in 16HBE cells gradually decreased,and these proteins can co-localize with viral VP1,redistribute in the nucleus and cytoplasm,and even degrade;(3)With the increase of virus infection days,there was no significant change in the expression of m6A methylation-related protein in ICR suckling mice and hSCARB2-transgenic mice.The results show that,in 16HBE cells,CVA16 and EVA71 infections had no significant effect on m6A methylation-related proteins,that is:CVA16 can also change the expression and cell localization of m6A methylation-related proteins in host cells.It is speculated that because m6A modification is cell-specific and contains multiple cell types in the rat brain tissue,it may be one of the reasons why there is no significant change in m6A methylation-related proteins in the rat brain tissue.The results of this study suggest that m6A modification may become the next potential target for enterovirus treatment.