The Role Of The N-linked Glycosylation In The PrM-E Protein Of JEV

Japanese encephalitis virus (JEV) is an important zoonotic pathogens. The virus dose not only damage the human nervous system and result in the death of adults and children, but also can cause reproductive disorders of swine. Japanese encephalitis occurs in South Asia, Southeast Asia and East Asia. In recent years, it was likely to be spread into Australia and Russia. The main prevention strategies of Japanese encephalitis are on vaccination, without specific therapeutic treatments for JEV infections.The N-linked glycosylation plays an important role in the processing and transport of viral proteins. Recently, more and more studies have demonstrated that the N-linked glycosylation has important biological functions in the virulence, replication, infection and immunity of the virus. The prM and E which are the improtant structural proteins of Japanese encephalitis virus can form prM-E heterodimer which play a major role in receptor binding, membrane fusion, neurovirulence and neuroinvasiveness and assemble into virus-like particles. The E protein can induce organism to produce neutralizing antibodies.The prM at Asn15 and protein E at Asn154 contain one N-linked glycosylation site, respectively. The N-linked glycosylation site in prM was highly conserved in the flavivirus, but the N-linked glycosylation site in E was variable. At present, the research on the N-linked glycosylation in Japanese encephalitis virus has not been investigated deeply.This research focused on the role of N-linked glycosylation site of prM and E on the folding and secreting of protein E, the forming of virus-like particle and virulence to the cells. The main research are showed as follows:1. Costructed the plasmids of the glycosylation-deficient prME Amplified the prME gene of Japanese encephalitis virus and then the gene was cloned into pcDNA3.1 (+). Generate three prME mutants with with glutamine substiting for asparagine through PCR mediated site-directed mutagenesis. Then mutant prME genes were cloned into the pcDNA3.1 (+), and constructed the plasmids:mcprME15, mcprME154 and mcprME15154. The results of sequencing showed the success of all mutation. Then these plasmids were transfected into HEK-293FT cells separately, and was proved to express the prM and E protein through the indirect immunofluorescence assays. The analysis of Western Blot showed that the mobility of N-glycosylation site-deficient prM and E protein were higher than the wild type protein in SDS-PAGE. It was confirmed that the N-linked glycosylation sites in prM and E proteins had been successfully eliminated.2. The role of N-linked glycosylation in the folding of E proteinThe pcprME, mcprME15, mcprME154 and mcprME15154 were transfected into HEK-293FT cells, respectively. The results of indirect immunofluorescence assays using the conformational antibody of E protein as primary antibody showed that the E protein which was expressed by mcprME15 and mcprME15154 didn't get its native conformation, when the N-linked glycosylation site was absent in prM protein. It indicated that the N-linked glycosylation site in prM was critical in the folding of E protein. When the N-linked glycosylation sites in E protein was absent, the most of the E protein was not properly folded and a little of E protein had got the correct conformation. It suggested that the N-linked glycosylation site in E protein played an important role in the folding of E protein. When it was ablated, the efficiency of folding of E was greatly reduced.3. Effect of N-linked glycosylation on the secretion of E proteinTransfected HEK-293FT cells with plasmids pcprME, mcprME15, mcprME154 and mcprME15154, and collected the supernatant., Detected the content of E protein in the supernatant by ELISA. The results showed that the content of E protein in pcprME was much higher than mcprME15, mcprME154 and mcprME15154. It indicated that secretion of E protein was greatly impaired when the N-linked glycosylation site in prM or E protein was absent.4. Effects of N-linked glycosylation on virus-like particles formation.Transfected HEK-293FT cells with plasmids pcprME, mcprME15, mcprME154 and mcprME15154 and then the cells were fixed with 2.5% glutaraldehyde. The virus-like particles of Japanese encephalitis virus which were formed by the prM and E protein had been observed in the cells which were transfected with pcprMEå’ŒmcprME154. The virus-like particles were not found in mcprME15,mcprME15154, even increasing the number of cell slices. It was suggested that the N-linked glycosylation site in prM was crucial for the formation of virus-like particles, while the N-linked glycosylatio site in E protein affected the efficiency of the formation of virus-like particles.5. Role of N glycosylation in prM-E proteins on the cytopathic effectHEK-293FT cells were transfected with plasmids pcprME, mcprME15, mcprME154 and mcprME15154 and harvested after digested with trypsin. The cells were stained with an Annexin V-FITC Apoptosis Detection Kit, according to the manufacturer's instructions. The results showed that the apoptosis rate of plasmid pcprME (9.38%) was higher than mcprME15 (2.00%), mcprME154 (3.74%) and mcprME15154 (1.30%). It indicated that the N-linked glycosylation had a role in the cytopathic effect which was caused by the prM and E protein.