Japanese Encephalitis Virus SfRNA Counteracts Cellular Interferon Response By Targeting The Translation Of PKR And IFITM2

Japanese encephalitis virus(JEV)is a single positive stranded RNA virus belonging to the flavivirus family flavivirus.Culex tritaeniorhynchus is the main carrier of JEV.Wading birds and pigs are natural hosts of infection for JEV,pigs are the main amplifying hosts of JEV in nature.Commercial pigs are inapparent infection mostly.Sows infected with JEV can cause miscarriage,boars infected with JEV can lead to decreased semen quality.JEV includes five genotypes:G?-G?.The G? type contains two branches,G?-a and G?-b.Each genotype has a different geographical distribution pattern.The geographical distribution of JEV genotypes has changed significantly in recent years.GI type gradually replaced G? type as the dominant strain in most of Asia,but the exact conversion mechanism is unknown.The production of subgenomic flaviviral RNA(sfRNA)by infected cells of members of the flavivirus family,including JEV,is a conserved biological phenomenon.Flavivirus 3'UTR contains Xrn-1 resistant elements(xrRNAs)that block the degradation of the viral genome by host exonuclease Xrn-1.Xrn-1 cannot completely degrade the viral genome,resulting in the production of sfRNA.JEV's 3'UTR consists of 4 stemp and loops(SLs),2 dumbbells(DBs),short hairpain(sHP),and 3' end stem loop(3'SL).In addition,there are short repeats(direct repeats,DRs),which include conserved sequences(CS)and repetitive conserved sequences(RCS).Pseudoknots(PKs)formed on the basis of SL-?,SL-?,DB-? and DB-? structures are crucial to the stability of xrRNAs.Therefore,four different subtypes of sfRNA can be formed theoretically.SfRNA is crucial to flavivirus life cycle,pathogenicity and host adaptability.Therefore,the study of JEV sfRNA is helpful to uncover the new mechanism of JEV infection host and provide reference for the prevention and control of JEV and the development of antiviral drugs.In this study,high-resolution Northern blot was firstly established to detect the difference of sfRNA subtype produced by JEV cells infected with G? and G? JEV Sequence alignment and phylogenyetic analysis were carried out on JEV sfRNA of NCBI published genotype.By detecting the expression of ISGs after JEV infection,it was found that JEV sfRNA could inhibit the cellular interferon response by targeting PKR and IFITM2 translation.The main research contents are as follows:1.Genotype I and III Japanese encephalitis virus sfRNA subtype identification and phylogenyeticanalysis.In order to detect JEV sfRNA subtype,a high-resolution Northern blot based on the Urea-Page was firstly established.The differences in sfRNA subtypes was detected by high resolution Northern blot in the infected cells of GIII JEV NJ08 strain,SA14-14-2 strain and GI JEV HN07 strain.It was found that JEV infected cells produced four subtypes of sfRNA,which were sfRNA 1-4 according to the fragment size.The subtypes of sfRNA produced by GI type and G? type JEV infected cells have the same characteristics,with the highest amount of sfRNAl,followed by sfRNA4,and the lowest amounts of sfRNA3 and sfRNA2.In order to further study the differences between GI and G? sfRNA,the sequence of JEV sfRNA of known genotypes published by NCBI was analyzed by multiple sequence alignment and phylogenyetic analysis.The results showed that the differences of JEV sfRNA sequences 10440,10673 and 10853 were genotypic(The locus was taken as an example by JEV HN07,GenBank:FJ495189.1).The change of 10477 nucleotides in GI was more favorable for base pairing in PK1 than GIII.The nucleotide similarity between genotype ? and type ? sfRNA groups is 79.2%? 100%.The nucleotide sequence similarity in GI sfRNA group was 81.6%? 100%,and that in G? sfRNA group was 85.6%? 100%.GI and G? JEV sfRNA nucleotide variation was dominated by base conversion,accounting for 70.7%and 73.8%of base mutations,respectively.Therefore,GI and G? JEV-infected cells produce sfRNA subtypes with the same characteristics,and sfRNA sequences within the same genotype are closely related.2.Japanese encephalitis virus sfRNA inhibits the expression of PKR and IFITM2.To investigate the biological function of sfRNA production in JEV infected cells,sfRNA production in JEV infected cells after 24hpi was detected by Northern blot.Western blot and qPCR showed that JEV infection down regulated interferon-stimulating proteins IFITM2 and PKR in the later stage,while not SFL proteins.This phenomenon is consistent with the time characteristics of sfRNA production.To investigate whether sfRNA plays a role in this process.JEV sfRNA was synthesized by in vitro transcription.After the transfection of sfRNA,the expression of IFITM2 and PKR proteins stimulated by IFN-a was decreased,while not the mRNA.In addition,deletion of the key SL-II region of sfRNA has been found to reduce its ability to affect the expression of IFITM2 and PKR proteins.The change of JEV sfRNA 10477 sites will make its inhibition of IFITM2 protein expression decrease,but it has no effect on the inhibition of PKR protein expression.Therefore,this study found that JEV sfRNA inhibits the expression of PKR and IFITM2 proteins during translation.In conclusion,JEV sfRNA can inhibit the expression of PKR and IFITM2 proteins during the translation stage,and the SL-II region is essential for the function of JEV sfRNA.The mechanism of inhibition still needs to be further explored.