Study On Interaction Of Japanese Encephalitis Virus NS4B And NS3 Helicase

Japanese encephalitis virus(JEV)belongs to the family Flaviviridae and genus Flavivirus,which is the cause of Japanese encephalitis.The infection of JEV can cause obvious symptoms of encephalitis.Pregnant sows may develop with high fever,abortion,stillbirth and mummified tires due to JEV infection,while boars develop with orchitis.Nowadays,JEV affects 25 Asian countries,where 60% population lives at risk of exposure to JEV.The public health problem due to JEV infection is still very serious.At the present,the forming of replication complex(RC)and precise molecular mechanisms for JEV RNA replication are still elusive.It's well known that all non-structure proteins of JEV are involved in the forming of replication complex,NS3 helicase unwinds to double stranded RNA.The NS4 B protein is the only non-structural protein which has a long fragment inside and outside the endoplasmic reticulum and plays a key role in the formation of flavivirus replication complex.In this study,we identified the sub-cellular localization and membrane topology of JEV NS4 B.Using coIP and GST pull down,and we confirmed the interaction between JEV NS4 B cytoplasmic loop and NS3 helicase.Importantly,we found that over-expression NS4 B and its cytoplasmic loop will influence the replication of JEV.The main results are as follows: 1.The interaction between JEV NS4 B and NS3 helicase.The JEV NS4 B gene and NS3 helicase gene were amplified by PCR firstly and then inserted into eukaryotic expression vector pcDNA3.1(+),pcDNA4.0,respectively.Next,we identified the expression of recombinant plasmid by indirect fluorescent antibody technique.HEK 293 T cells were co-transfected with the recombinant plasmid.After 36 h,cells were collected and the interaction between NS3 helicase and NS4 B protein was identified by coimmunoprecipitation.The result suggested that NS4 B actually interacted with NS3 helicase in the absence of the other viral proteins.2.Identification of the cytoplasmic loop of JEV NS4 B protein.To establish a membrane topology model of NS4 B,we analyzed the amino acid sequence of JEV NS4 B by using multiple software packages.Five hydrophobic regions was predicted in the NS4 B protein.In order to determine the regions of NS4B's trans-membrane domains,we amplified the fragments encoding different peptides of NS4 B and cloned into eukaryotic expression vector pEGFP-C1.We transfect the recombinant plasmid to Hela cells,and detected the sub-cellular localization by IFA.The result demonstrated that TMD1 and TMD2 don't span the membrane but ruther reside in the ER lumen.The amino acids 118-175 of NS4 B were cytoplasmic loop,which is most likely to be involved in the interaction of NS3.3.The interaction between JEV NS4 B cytoplasmic loop and NS3 helicase.The JEV NS4 B cytoplasmic loop gene was amplified by PCR and then inserted into eukaryotic expression vector pEGFP-N1.In combined with pcDNA3.1(+)-NS3 helicase,the recombinant plasmids were co-transfect to HEK 293 T cells.We performed a co-IP experiment using the cell lysates.The result demonstrated that NS3 helicase interacted with NS4 B cytoplasmic loop.The JEV NS4 B cytoplasmic loop gene was amplified by PCR and then inserted into prokaryotic expression vector pGEX-6p-1.The soluble GST-NS4B(118-175)protein can be obtained through transforming the constructed expression plasmid pGEX-6p-1-NS4B(118-175)into BL21 E.coli and induced for 20 hours at 16? with 0.5 mM IPTG.By the GST column affinity chromatography purification,the pure NS4 B cytoplasmic loop was acquired.And by the Nickel column affinity chromatography purification,the pure NS3 helicase protein was acquired.The direct interaction between NS4 B and NS3 helicase was verified by GST pull down.The result demonstrated that the NS3 helicase and NS4 B cytoplasmic loop can interact with each other.4.The influence of NS4 B and its cytoplasmic loop on JEV replication.During the replication of JEV,NS4 B locates the NS3 helicase to the endoplasmic reticulum(ER)interacted with the NS3 helicase.Importantly,both NS4 B and NS3 helicase participate in the formation of viral replication complex(RC).We transfect the recombinant plasmid pEGFP-N1-NS4B(118-175)and pEGFP-N1-NS4 B to Hela cells,and infected the cells with JEV after 24 h.The effect of NS4 B on the replication of JEV virus was verified by plaque assay.The result demonstrates that the over-expression of NS4 B and NS4 B cytoplasmic loop have little influence on JEV replication,but this inhibition need further confirmation.