Screen Of A Membrane Protein Molecule From Vero Cells Capable Of Binding Japanese Encephalitis Virus(jev)

Japanese encephalitis virus,JEV,the cause of central nervous system infection, i.e., Japanese encephalitis(JE) which has a mortality rate of 10%,is an encapsulated positive strand RNA virus, belonging to the Flavivirus. This virus is a great threat to public health. In recent years, pandemic JEV has been spreading and posses a serious problem to tropical and semi-tropical countries. With the looming of the possible use of this virus as a bio-weapon, the importance of this virus has been addressed again.Although we have a well-organized system in the prevention, diagnosis and treatment of JE, we have few knowledge of how the virus infects cell, and of the receptor for JEV, which is vital in the attachment of virus to host cells.The interaction between the virus and corresponding virus receptor on host cells is a pivotal step in the infection process of the virus. Thus, the importance of the study of virus receptor resides not only in the mechanism of this interaction itself, but also in the typing of virus, and even more, in the development and screening of new drugs and vaccines. Therefore, we hope to find the candidate receptor of JEV on Vero cells, out of which authentic receptor of JEV may be identified.In the present study, the membrane protein of Vero cell was extracted with buffer containing nonionic detergent NP-40 under mild condition and the fresh extract was co-immunoprecipitated with JEV. The precipitates, lysates from Vero cell membrane protein, and JEV were analyzed by SDS-PAGE. The bands was compared, and three distinct bands with molecular weight of 90, 45 and 34 kDa respectively, were cut and analyzed by mass spectrometry(MS). The data obtained by MS was matched against a protein library. The MS reports suggested that the 90 kDa protein was Heat Shock Proteinβchain and the 45 and 34 kDa protein are unnamed protein products. The identity of the candidate receptor protein was then verified by flow-cytometry(FCS)(Results were expressed as mean±SD). The results showed that the background of Vero cell was 1.64±0.06 and that the infection rate of JEV(MOI 1)to Vero cell was 89.9±0.72, which fell to 79.91±1.90(P<0.05), and futher to 68.28±2.79(P<0.05), if HSP90βantibody was added at 1:50 and 1:10, respectively. These results showed that anti-HSP90βmonoclonal antibody significantly prevented the attachment of JEV to Vero cell. The same results were also obtained with immunofluorescent microscopy in the virus blocking assay.The results listed above suggested that HSP90β, which is normally expressed on the surface of Vero cells, can bind JEV. More work are need to certify HSP90βas the natural JEV receptor.