Effects Of Sec62/Sec63 Complex On Replication Of Japanese Encephalitis Virus

Japanese encephalitis JE is a mosquito-borne zoonosis caused by the Japanese encephalitis virus.JEV is a member of the Flaviviridae Flavivirus family.JEV can infect people and a variety of animals,most of them are recessive infection,human and horse infection will cause neurological symptoms and encephalitis,serious can lead to death.The main vector of transmission is culex tritaeniorhynchus,which is transmitted from host to host through mosquito bites.Waterbirds and pigs are the main extended hosts and are the terminal hosts.At present,Japanese encephalitis still have no effective treatment,mainly to prevent by vaccination.Although Japanese encephalitis has been effectively controlled,there are still many gaps in the study of the replication cycle of Japanese encephalitis virus.Sec62/Sec63 complex is composed of Sec62 protein and Sec63 protein,which is a transmembrane protein in the endoplasmic reticulum.The main function is transporting some proteins between the Sec61 channel.Sec62 can interact with ribosomes to coordinate protein translation in ER,and further influence the cellular response induced by endoplasmic reticulum stress.Sec63 is composed of three transmembrane domains.The J domain interacts with the molecular chaperone Bip in the endoplasmic reticulum to promote translocation of precursor proteins through the Sec61 channel.In order to study the effect of Sec62/Sec63 complex on the replication of JEV,HEK-293 cell lines with SEC62 or SEC63 gene knockout were prepared by CRISPR/Cas9 genome editing technology.After identification by sequencing and western blot,two cell lines were successfully constructed and named as SEC62 KO HEK-293 and SEC63 KO HEK-293 cells.After infection HEK-293,SEC62 KO HEK-293 and SEC63 KO HEK-293 cells with JEV on MOI of 1 and 0.05,the efficiency of JEV replication was detected by indirect immunofluorescence test.The results showed that the number of cells with fluorescence in both SEC62 KO HEK-293 cells and SEC63 KO HEK-293 cells infected by JEV at different doses were lower than that in HEK-293 cells.After infecting HEK-293,SEC62 KO HEK-293 and SEC63 KO HEK-293 by JEV on MOI=1,western blot results showed that the expression of JEV E protein in the cells of SEC62 KO HEK-293 and SEC63 KO HEK-293 could not be detected at 12 h,24h or 48 h,while the expression of JEV E protein in the cells of HEK-293 increased.Virus titers were determined after infecting the above three cell lines by JEV on MOI=1,0.5 and 0.05,respectively.The results showed that there were no difference in virus titers between the SEC62 KO HEK-293,SEC63 KO HEK-293 cells and the HEK-293 cell group at 12 h after infection(p>0.05),while the difference was significant at 24 h and 48 h after infection.Virus titers in supernatant of SEC62 KO HEK-293 and SEC63 KO HEK-293 cells were significantly lower than those in HEK-293 cells(p<0.01).The above results showed that both SEC62 and SEC63 genes knockout inhibited JEV replication.This study provides a reference for further exploring the mechanism of host factors such as Sec62 and Sec63 impacting on JEV replication.