Effect Of Host Protein DAZAP2 On The Proliferation And Replication Of JEV

The NS1 protein of Japanese encephalitis virus(JEV)is an important non-structural protein,and plays an important role in the proliferation,replication,assembly and immune evasion of viral life cycle.Our previous study found that host protein DAZAP2 interacted with the NS1 proteins.Since the roles of host protein in JEV replication has indicated,we have a strong interest in exploring how participate in JEV replication.First,DAZAP2 gene targeting interference plasmid and the DAZAP2 gene knockout cell line were constructed.The fluorescence quantitative standard curve of NS1 gene,E gene and?-actin for detecting the transfection level,DAZAP2 gene expression,and JEV mRNA level were successfully constructed.R~2 was greater than 0.99.The DAZAP2 gene targeting interference plasmid were successfully constructed and the maximum interference efficiency was about53.7%.The DAZAP2 gene knockout cell line were successfully constructed by CRISPR/Cas9.Then,DAZAP2 protein expression was inhibited by shRNA and CRISPR/Cas9 and found that positively regulated JEV mRNA and progeny virus particle levels.After interfered with the expression of the host protein DAZAP2 on 293 cells,relative fluorescence quantification and Western Blot(WB)detection showed no significant changes in JEV proliferation replication.The relative fluorescence quantification and WB were executed after the JEV infected the DAZAP2 knockout cell line.The JEV mRNA level was found to be approximately 1.6-fold higher,and the progeny virus titer was 6.35 pfu/ml,which was 1.27times that of the control group.However,the DAZAP2 knockout cell line was transfected with the Japanese encephalitis virus replicon,and no obvious changes were discovered.Ultimately,over-expression DAZAP2 protein was found have affection on JEV mRNA levels.It's was verified that the JEV mRNA had significant difference and it was increased about 3 times compared with the control group after overexpression the host protein DAZAP2.In summary,our data demonstrate that DAZAP2 play an important role in propagation and replication of JEV.