Effects Of Host HMGB1 Protein On The Proliferation Of Japanese Encephalitis Virus In Huh7 Cells

The pathogen of Japanese encephalitis(JE)is Japanese encephalitis virus(JEV)which is a member of the Flavivirus genus,Flaviviridae family.Japanese encephalitis is mainly prevalent in some countries and regions of Asia and the Western Pacific.The research is relatively absent and so that the life cycle of JEV and the pathogenesis of JEV are still unclear.But JEV is a zoonotic virus that can't be ignored.It is mainly transmitted by Culex tritaeniorhynchus.And it can cause abortion in pregnant sows,stillbirth of dead or weak offspring,orchitis in boars and so on.There are more than 3 billion people live in areas vulnerable to JEV,and approximately 67 000 reported cases and 20 000 deaths each year.Among them,30 to 50 percent of survivors of JEV infection have permanent neurological sequelae.It is necessary to understand the pathogenesis of JEV.During the life cycle of JEV,there exists interaction with many host proteins.Analysing the relationship among them is very essential to explore the life cycle of JEV,to provide antiviral drugs and to control the spread of JEV.In this study,BJB strain of JEV was used as experimental object.The study observed that the expression of host protein high mobility group box 1(HMGB1)decreased significantly in Huh7 after infection.HMGB1 protein is a kind of DNA binding protein,which widely exists in nucleus and cytoplasm.It plays an important role in inflammation,immunity,cell growth,proliferation and death.HMGB1 in the cytoplasm also binds to various proteins and participates in autophagy and cancer progression.In this study,the eukaryotic expression vector,pRE-HMGB1-Strep,was constructed and overexpressed HMGB1 protein in Huh7 cells.The study found that the proliferation of BJB was inhibited when we increased the expression of HMGB1 protein.Meanwhile,after silencing HMGB1 protein in Huh7 cells by RNAi assay,the RNA level and the titer of BJB virus increased significantly.Then we transformed the Huh7 cells through the CRISPR/Cas9 system.This study got the cell line KO-HMGB1-I which can not express HMGB1 protein.It was found that the proliferation of BJB increased obviously in KO-HMGB1-I,and it was consistent with the RNAi assay.Therefore,we can conclude that the overexpression of HMGB1 protein in Huh7 cells can inhibit the replication and proliferation of BJB virus,while the silencing of HMGB1 protein can promote the replication and proliferation of the virus.Some studies have shown that the increase of HMGB1 expression in cytoplasm induces autophagy and the increase of autophagy degree inhibits the replication of JEV.In this study,it was found that the mRNA level of late glycation end product receptor(Receptor for advanced glycation end products,RAGE was inhibited in Huh7 after infection.However,the increased expression of HMGB1 could restore the expression of RAGE.It has been shown that the increase of RAGE mRNA level can indirectly induce autophagy through interaction with HMGB1.Therefore,this study speculated that HMGB1 may affect cytokines and autophagy by affecting the expression of RAGE,and ultimately affect the replication and proliferation of BJB in Huh7.