Mutations at KFRDI and VGK domains of enterovirus 71 3C protease affect its RNA binding and proteolytic activities

The 3C proteases (3Cpro) of enterovirus 71 (EV71) is a good molecular target for drug discovery. Notably, this protease was found to possess RNA-binding activity. The regions responsible for RNA binding were classified as ‘KFRDI’ (positions 82–86) and ‘VGK’ (positions 154–156) in 3Cpro by mutagenesis study. Although the RNA-binding regions are structurally distinct from the catalytic site of EV71 3Cpro, mutations in the RNA-binding regions influenced 3Cpro proteolytic activity. In contrast, mutations at the catalytic site had almost no influence on RNA binding ability. We identified certain mutations within 3Cpro which abrogated both the RNA-binding activity of the expressed, recombinant, protease and the ability to rescue virus from an infectious full-length clone of EV71 (pEV71). Interestingly, mutation at position 84 from Arg(R) to Lys(K) was found to retain good RNA binding and proteolytic activity for the recombinant 3Cpro; however, no virus could be rescued when pEV71 with the R84K mutation was introduced into the infectious copy. Together, these results may provide useful information for using 3Cpro as the molecular target to develop anti-EV71 agents.