Using RNAi to investigate the role of V-ATPase in C6/36 cells infected by Japanese encephalitis virus

• Main Authors: Shu-Ting Wang, 王淑婷
• Other Authors: Wei-June Chen
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/28377816055838571799

碩士 === 長庚大學 === 基礎醫學研究所 === 94 === Japanese encephalitis is an important arboviral disease in Asia. It is caused by Japanese encephalitis virus (JEV) infection. The JEV is a member of the virus family Flaviviridae, which is transmitted to human through the bite of mosquitoes. In addition to mosquito cells, JE virus also infects mammalian cells. Many researches have reported that JEV enters mammalian cells by receptor-mediated endocytosis. In 1998, Nawa treated mosquito cells with bafilomycin A1, a specific inhibitor of vacuolar type H+-ATPase (V-ATPase) and observed inhibited growth of JEV in the cells. These results indicate that JEV may also enter C6/36 cells through the endocytic pathway involving intracellular acidic compartments. In this study, we first designed specific siRNAs to inhibit V-ATPase of mammalian cells (BHK-21). The results indicated that siRNA may reduce V-ATPase mRNA expression as well as virus replication. This study also demonstrated that the V-ATPase plays a role in the JEV infection to mammalian cells. Subsequently, we used dsRNAs to knock down the V-ATPase expression of C6/36 cells based on the hypothesis that JEV takes the same route to enter the mosquito cells as does in mammalian cells. The results showed that the dsRNA could inhibit the expression at both mRNA and protein levels of the V-ATPase and reduce virus replication as well. These results have revealed that JEV actually enters mosquitoe and mammalian cells by the V-ATPase-regulated endocytosis. In addition, functional to inhibition of V-ATPase by RNAi also blocks the JEV replication in mosquito cells. Results shown in this study have provided a promising way for the control of JEV infection in future.