RNA Binding Activity of Enterovirus 71 3C Protease

• Main Authors: Chiayn Chiang, 蔣佳穎
• Other Authors: Shin-Ru Shih
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/87599281987367436683

碩士 === 長庚大學 === 基礎醫學研究所 === 91 === The 3C protease of enterovirus 71 is a good molecular target for drug discovery. This protease was also found to bind to the cloverleaf structure in the 5’-noncoding region of its own viral RNA. Mutagenesis study of the KFRDI region (position 82 to 86) and VGK region (position 154 to 156) was proved that these regions were responsible for binding of 3C protease. Although the catalytic site of 3C protease is structurally distinct from the RNA binding regions, mutations in RNA binding regions affected its proteolytic activity. However, mutations at the catalytic site had no effect on RNA binding ability. Using the transcript of infectious clone (pEV71) at mutations in KFRDI and VGK regions to investigate the effects of RNA binding activity on virus replication. We found that certain mutations that failed the RNA binding activity also impaired the negative-stranded viral RNA synthesis. And mutations in KFRDI and VGK regions of pEV71 other than K82Q, D85N, V154T, and K156Q cannot form plaque. These results showed that RNA binding activity of 3C protease was correlated with both viral RNA synthesis and virus growth. Interestingly, R84K of the recombinant 3C protease was found to retain good RNA binding and proteolytic activity. And the corresponding mutation in pEV71 exhibited viral RNA synthesis in EV71-RNA transfected cells. However, pEV71 with R84K mutation could not form plaque, implying that this site in 3C protease may possess another function essential for plaque formation. Furthermore, R84K mutant can interfere with the growth of wild-type Enterovirus 71 when it was over expressed in cells. Together, these results can help to clarify the role of 3C protease in viral replication and provide useful information in drug development using 3C protease the molecular target.