Screening And Identification Of Host Protein Cleaved By Japanese Encephalitis Virus NS2B/3 Protease

Japanese encephalitis virus(JEV)is a mosquito-borne flavivirus that causes viral encephalitis in humans and reproductive disorder in pigs.Currently,there is no specific therapy for JEV,and the main prevention and control strategy is prevention through vaccination.The JEV NS2B/3 protease cleaves viral polyproteins and their sites have been sporadically reported and validated,but the host proteins and sites cleaved by it have not been reported,and there is currently a lack of a screening method that can be used for protease cleavage of host proteins.Based on this issue,we established a method to screen for JEV NS2B/3 protease cleaved host proteins,and obtained the cleaved host proteins,and initially investigated their effects on proliferation of JEV.The specific results of the study are as follows:1.Screening and identification of JEV NS2B/3 protease cleaving host proteinUsing the method of protein N-terminal omics,the primary amine(α-NH2)of the protein was biotin-labeled using a biotin labeling kit,and then streptavidin magnetic beads were used to capture the labeled protein,and JEV NS2B/3 protease was incubated with magnetic beads to collect and concentrate the incubated elated protein for LC-MS/MS analysis.Finally,the host protein cleaved by the NS2B/3 protease is obtained.In this study,a total of 153 proteins that could be cleaved by JEV NS2B/3 protease were screened.GO analysis showed that more than 70% of the screened proteins were located in the cytoplasm,in terms of molecular function,more than 80% of the proteins were involved in protein binding or RNA binding.KEGG signaling pathway analysis showed that the proteins involved in translation were the most,followed by the proteins involved in signal transduction.Combining the function of the protein and the mass spectrometry scoring results,the protein was selected and verified to be cleaved.Finally,DDX5 protein was identified to be cleaved by JEV NS2B/3 protease.2.Cleavage of DDX5 protein by JEV NS2B/3 proteaseDDX5 was co-transfected with eukaryotic plasmid of NS2B/3 protease into HEK-293 T cells,and it was verified by Western Blot that DDX5 could be cleaved by NS2B/3 protease in vivo and its cleavage was dose-dependent.JEV NS2B/3 protease is a serine protease,and to verify whether the protease activity affected DDX5 cleavage,the eukaryotic plasmid of the protease of the JEV NS2B/3 mutants was co-transfected with DDX5.The results showed that only the wild-type protease could cleave DDX5 while the protease of the JEV NS2B/3 mutants lacking the protease activity could not.Therefore,it can be concluded that the cleavage of DDX5 is dependent on the serine protease activity of JEV NS2B/3 protease.3.The JEV NS2B/3 protease cleavages DDX5 at R502Based on the characteristics of the substrate site cleaved by JEV NS2B/3 protease and the fragment size produced by DDX5 cleavage,it was hypothesized that the specific site cleaved by DDX5 was either R502 or R524.By constructing mutants R502 A and R524 A to validate their cleavage,only the R502 A mutant was found not to be cleaved.Therefore,it was possible to determine that the R502 is the only JEV NS2B/3 protease cleavage site on DDX5.4.Effect of DDX5 protein on JEV replicationIn order to study the effect of DDX5 on JEV replication,DDX5 was overexpressed and silenced in HEK-293 T cells,and then infected with JEV P3 strain.Western Blot was used to detect DDX5 expression,q RT-PCR was used to detect JEV RNA replication level and intracellular interferon changes,and plaque assay was used to detect virus titers.The results showed that the silencing of DDX5 could promote the production of interferon and inhibit the replication of virus.Overexpression of DDX5 inhibits interferon production and promotes viral replication.5.DDX5’s cleavage facilitates JEV replicationTo investigate the effect of cleavage of DDX5 on JEV,eukaryotic plasmids of cleavage fragments of DDX5 were constructed.The results of q RT-PCR and plaque assay showed that the promotion of JEV by R502 A was significantly lower than that of DDX5,1-502aa-DDX5 and 503-614aa-DDX5,which proved that the cleavage of DDX5 was beneficial to the replication of JEV.