Mechanism Of Japanese Encephalitis Virus Helicase Degradation Of Host MiRNA

Flaviviridae is a family of viruses which consists of a positive-sense singlestranded ribonucleic acid(RNA)genome can cause a variety of important human and animal infectious diseases.Flaviviridae mainly spread through arthropod vectors,such as mosquitoes and ticks.Major members of the Flaviviridae family include Japanese encephalitis virus(JEV),West Nile virus(WNV),Zika virus(ZIKV),dengue virus(DENV),and hepatitis C virus(HCV)which remain a major global public health threat.Previous study reported that eukaryotic miRNAs play an important role in the replication and propagation of viruses.Expression or targeting of host miRNAs can be involved in cellular antiviral responses.Generally,host miRNAs involved in viral lifecycles promote or suppress infection through complex regulatory pathways.miRNAs can also be encoded by the viral genome and expressed in host cells,which have a common sequence or completely different sequences with the host miRNA.Therefore,how the Flaviviridae virus regulates the expression and function of host miRNAs,which plays an extremely important role in the occurrence and development of diseases.There are many studies on the regulation of miRNA biogenesis by flavivirus,but the modulation of miRNA biogenesis by helicase of NS3 in flavivirus has not been reported.Firstly,we use high-throughput sequencing to detect the expression of miRNA in mouse brain tissue and neuronal cells after infected with Japanese encephalitis virus(JEV),the result showed that a global down-regulated of host miRNAs.We detect the expression of host miRNA after treated by transcriptional inhibitors Favipiravir(T-705),?-Amanitin and translation inhibitor cycloheximide(CHX)and found that the degradation of host miRNA was due to the JEV transcript.Since JEV transcript did not suppress host miRNA,to further determine which viral protein were sufficient to confer the host miRNA degradation,10 pcDNA3.1 expression vectors encoding each structure protein(E,C and PrM)non-structure protein(NS1,NS2 A,NS2B,NS3,NS4 A,NS4B and NS5)of JEV were constructed.The miR-466d-3p expression was measured 48 hpi,only NS3 induced a significant decrease of mature miR-466d-3p.Since the JEV NS3 represented a critical role in turnover of host miRNA,we constructed pcDNA3.1 expression vector encoding NS3 of ZIKV,WNV,DENV1,DENV2,CSFV and HCV to further identify whether the helicase of other flavivirus could degrade the host miRNAs.These results indicated that the most of NS3 of Flavivirus decrease the expression level of miR-466d-3p.Furthermore,we use siRNA and two chemical synthesis inhibitors(5,6-dichloro-1-?-D-ribofuranosyl-1H-benzimidazole,DRB and Ivermectin,IVER)of NS3 helicase to assay the expression level of miR-446d-3p.The result showed that block the function of NS3 helicase can significantly suppress degradation of miR-466d-3p.As we all known that blocking the activity of the RISC silencing complex proteins Dicer and Argonaute can inhibits the production of miRNAs.We used qRT-PCR to detect the gene expression of major constituent protein in RISC.There is no influence of the gene expression level of major constituent protein in RISC after infection with JEV.Secondly,the results of the helicase blocking and miRNA double-stranded unwinding assay in vitro indicate that NS3 can lead to the unfolding of the doublestranded structure of pre-miR-466 d and abnormal miRNA function.We also use computational models and RNA immunoprecipitation assay which found that argininerich domains of NS3 are critical for pre-miRNA binding and the degradation of host miRNAs.Importantly,site-directed mutagenesis of conserved residues on NS3 revealed that R226 G and R202 W significantly reduced the binding affinity and degradation of pre-miR-466 d.Together,these results extend the helicase of Flavivirus function beyond unwinding duplex RNA to the decay of pre-miRNAs,which provides a new mechanism of NS3 in regulation of miRNA pathways and Antiviral immune response.