The Affection Of NS3 Protein To Proliferation Of Japanese Encephalitis Virus And Screening For The Interacting Protein In Host Cells

Japanese encephalitis is a zoonotic, mosquito-borne infectious diseases caused by Japanese encephalitis virus ?JEV?. JEV infections can cause great harm to human health and the breeding industry. The complete cycle of JEV infection contains adsorption, uncoating, synthesis, assembly and release. The nonstructural protein NS3 of JEV is a multifunctional protein, which plays a key role in the process of viral infection and reproduction. The structure of JEV is relatively simple, so the interactions between viral protein and cellular protein are considered to be an important mechanism for viral infection. In this study, the RNA interference was used to determine the affection of NS3 to viral replication in vivo and in vitro. To explore the functional mechanism of NS3 for the JEV life cycle, the yeast two-hybrid system and co-immunoprecipitation test were used to screen the host cellular protein which interacting with NS3. Our study will not only push the research of mechanism of JEV infection, but also bring new strategy to seek the antiviral drug targets for JEV.1. The affection of NS3 for JEV replicationBase on the JEV NS3 gene, we designed and synthesised 4 RNAi target sites ?genome 4656-467,4827-4847,4916-4936 and 5361-5381?. The target sites were inserted into the expression vector pGPU6/GFP/Neo to construct the pGP-shRNA which could introduce shRNA into cells. The suppression efficiency of pGP-shRNA to NS3 gene was confirmed. The pGP-shRNA were transfected into BHK21 cells which were infected JEV after 24h, then the virus cultures were collected 72h post-infection. To detect the change of viral replication, fluorescence quantitative PCR, plaque reduce experiment, indirect immunofluorescence and western blot were used in our study. The results showed the 4 shRNA expression plasmids could inhibit JEV replication in varying degrees. Among these, pGP-NS3-4 plasmid ?genome 5361-5381? was most efficient, which reduced 93.9% of the JEV mRNA and lowered virus titer approximately 950-fold. The results of indirect immunofluorescence and western blot showed JEV replication was clearly inhibited in BHK21 cells.The 7-days-old suckling mice were equally divided to 7 groups ?n=6?, and the four shRNA expression plasmids were inoculated into suckling mice by intracranial injection. Then the mice wree challenged with JEV of 10×LD₅₀ dose after 24h. The suckling mice were sacrificed five days after challenge and the brains were excised and homogenized in DMEM to produce 10%?w/v? suspensions which were tested by plaque reduction experiment to measure the virus titer. The results showed 4 plasmids could obviously inhibit JEV replication in suckling mice brain, especially pGP-NS3-4 plasmid reduced virus titer about 2400-fold. In addition, we inoculated the pGP-NS3-4 plasmid into 3-weeks-old mice, which were challenged with JEV with the different dose (10×LD₅₀ and 50×LD₅₀) after 24h. The result showed that pGP-NS3-4 plasmid could protect 3-weeks-old mice from the lethal induced by JEV. The 70% of the mice challenged with 10×LD₅₀ were protected by pGP-NS3-4, and 60% of mice challenged with 50×LD₅₀ JEV survived.The results of experiment in vitro and in vivo revealed that when NS3 gene was silenced, the JEV replication activity will reduced markedly. This study proved the NS3 protein is required for replication of JEV. In addition, we found an efficient target site of NS3 gene silencing, which could observably suppress the JEV replication.This study push the research of the function of NS3 protein and antiviral drug targets for JEV.2. Screening of the host cell interaction proteins for JEV NS3 proteinThe NS3 gene of JEV was amplified according to the sequence of wild isolates SCYA201201, and the NS3 gene was inserted into the pGBKT7 vector to construct pGBKT7-NS3 bait plasmid.Then the bait plasmid was transfered into yeast strain Y2HGold to construct the bait yeast. The bait yeast was identified with some tests including the toxicity, activation authentication and target protein expression. The results showed the insert of NS3 was no toxic effection to bait yeast, and NS3 gene had no autoactivation and the bait yeast could expression NS3 protein exactly.The bait yeast and human brain tissue cDNA library yeast were mixtured for the yeast two hybrid on the Gold Yeast Two Hybrid System. The positive clones were screened on DDO/X/A and QDO/X/A lacked nutrition medium. After three rounds of screening, there were 21 positive clones were acquired. The plasmid of positive clones were extracted, and the monoclonals were picked and library plasmids were extracted to sequencing and analysis after the transformation of Escherichia coli DH5?. The results showed that, there were eight repeats, four non-coding regions and one frameshift mutations sequences, eventually eight host interaction proteins coding gene were acquired. The eight sequences were maked to the Blast analysis on NCBI, and the results showed the similarity between the eight sequences and reference sequences of Genbank were all above 99%. The novel eight host interaction proteins respectively are FBLN5. PPP2CB, CRBN, GPR137B. UBE2N, ZNF350, Hsp40 and COP9. Furthermore, the eight library plasmid were transformated into yeast strain Y187 to construct the prey yeast. The prey yeast and NS3 bait yeast were mixtured for reply yeast hybrid, meanwhile the prey yeast mixtured with mock bait yeast were set up to the activation control group. The results of the eight groups were all positive, and that of the activation control group was negative, the results of reply yeast hybrid indicated the eight host interaction proteins are the candidate interactors of JEV NS3 protein.This study provides a good foundation for the further research of the interaction networks of NS3 protein and the mechanism of NS3 protein participate in JEV replication.3. Identification of the interaction between NS3 and the candidate interactorsIn this study, the co-immunoprecipitation test was done to further verify the interaction of eight host interactors with NS3 protein. Firstly, the total RNA were extracted from human kidney epithelial cells ?293 cell? reverse transcribed into cDNA. The coding region of the eight genes were amplified base on the cDNA, and the eight genes were respectively inserted into the eukaryotic expression vector pCMV-HA. Meanwhile, the NS3 gene was inserted into the pEGFP vector to construct the eukaryotic expression plasmid pEGFP-NS3. These plasmids were transfected into 293 cells respectively for expression, and results of Western blot showed six host protein and NS3 protein had been successfully expressed.The NS3 expression plasmid and interactor expression plasmid were combined one-to-one, and co-transfected into 293 cells for expression, the cells were nondenaturing dissociated to collect protein 24h after transfection.The protein interactions complexes were coprecipitated by immunomagnetic beads, and the collection were loaded to SDS-PAGE and detected by Western blot. The results showed that the heat shock protein Hsp40 ?DNAJB6? of host cell could combined with the JEV NS3 protein in 293 cells, which demonstrated that Hsp40 ?DNAJB6? protein is the host interactor of JEV NS3 protein.This study firstly dmonstrated that the host cell heat shock protein Hsp40?DNAJB6? in host cell is the interacting protein of JEV NS3 protein. This study provides important support for the further research of the mechanism of NS3 protein mediated JEV replication.