Effect Of Host Proteins Of HSP70 And ZBTB-38 On The Proliferation Of Japanese Encephalitis Virus

Japanese encephalitis virus(JEV)is the causative agent of Japanese encephalitis(JE),which is generally regarded as an importanzoonotic disease.Porcine is the intermediate amplified host.JEV can infect hosts through a variety of pathways.Our previous study has shown that JEV can replicate in the lungs of mice and cause respiratory tract transmission,but the molecular mechanism of JEV leading to respiratory infections is still unclear.So that,we focused on the interaction of JEV with host respiratory cells,explored the interacting proteins between JEV and porcine alveolar macrophages(PAM)and verified its effect on JEV proliferation.In this study,the co-immunoprecipitation assay was firstly used to screen the host proteins of PAM interacting with JEV and the HSP70 and ZBTB-38 proteins were successfully screened by LC/MSMS.Sencondly,we constracted the HSP70 and ZBTB-38 knockout cell lines by CRISPR/Cas9 technology,and the knockout effect was detected by DNA sequencing and indirect immunofluorescence.The sequencing results showed that the DNA sequences of both two genes were successfully frameshift,and the indirect immunofluorescence was no specific fluorescence,which was indicated that HSP70 and ZBTB-38 were successfully knocked out.Then,the mRNA of JEV levels decreased by 2.3-fold and 9-fold 48 hours post infection(h.p.i)of JEV in the knockout cell lines PAM.KO.HSP70 and PAM.KO.ZBTB-38,and the viral protein expression levels also reduced 0.25 and 1 times,respectively.What's more,the virion numbers were 0.3 times lower than the control group in 72 h.p.i.Finally,the HSP70 and ZBTB-38 genes were respectively overexpressed in the knockout cell lines PAM.KO.HSP70 and PAM.KO.ZBTB-38,which were infected JEV after 48 h.On the contrary,it showed that the mRNA of JEV levels increased by 0.9-fold and 1.5-fold and the viral protein expression levels also reduced 8.5 and 0.5 times,respectively.The virion numbers were enhanced obviously than the control group.Therefore,this study confirmed that the host proteins HSP70 and ZBTB-38 are JEV interacting proteins and positively regulate the proliferation of JEV.This rusults will provide preliminary research data for the study of pathogenic mechanism of JEV,especially the respiratory transmission caused by JEV infection.