Blocking human enterovirus 71 replication by targeting viral 2A protease

Objectives: Human enterovirus 71 (EV-71), a member of the Enterovirus genus, constitutes a major public health issue in the Asia-Pacific region, where it is associated with several severe neurological complications. There is currently no effective vaccine or antiviral against EV-71. The aim of this study was to determine whether the six amino acid peptide LVLQTM, which was previously shown to inhibit human rhinovirus (HRV) 2A protease (2Apro) activity in vitro and HRV replication in vivo in mice, could be of more general use against enteroviruses and more particularly against EV-71. Methods: To investigate whether the LVLQTM peptide was a pseudosubstrate of EV-71 2Apro, a recombinant luciferase containing the LVLQTM sequence was designed so that recognition of this sequence by 2Apro led to luciferase activation. Direct interaction between EV-71 2Apro and the LVLQTM peptide was further confirmed by isothermal titration calorimetry. We then tested the effects of the peptide on EV-71 2Apro cleavage activity and EV-71 replication in HeLa cells. Results:We showed that the LVLQTM peptide behaved as an effective substrate analogue of EV-71 2Apro, which binds into the active site of the protease with a dissociation rate constant of 9.6 mM. Moreover, LVLQTM signifi