Defining substrate selection by rhinoviral 2A proteinase through its crystal structure with the inhibitor zVAM.fmk

Highlights: • We show that rhinovirus 2A ^pro cleave GAB1. • RV 2A ^pro from genetic groups A and B cleave GAB1 at different sites • RV 2A^pro from genetic groups A and B cleave equally well when P1 is methionine • RV-A2 2A^pro zVAM.fmk inhibitor complex shows two positions for methionine • Hydrophobic pocket at P4 appears conserved in all enteroviral 2A^pro Picornavirus family members cause disease in humans. Human rhinoviruses (RV), the main causative agents of the common cold, increase the severity of asthma and COPD; hence, effective agents against RVs are required. The 2A proteinase (2A^pro), found in all enteroviruses, represents an attractive target; inactivating mutations in poliovirus 2A^pro result in an extension of the VP1 protein preventing infectious virion assembly. Variations in sequence and substrate specificity on eIF4G isoforms between RV 2A^pro of genetic groups A and B hinder 2A^pro as drug targets. Here, we demonstrate that although RV-A2 and RV-B4 2A^pro cleave the substrate GAB1 at different sites, the 2A^pro from both groups cleave equally efficiently an artificial site containing P1 methionine. We determined the RV-A2 2A^pro structure complexed with zVAM.fmk, containing P1 methionine. Analysis of this first 2A^pro-inhibitor complex reveals a conserved hydrophobic P4 pocket among enteroviral 2A^pro as a potential target for broad-spectrum anti-enteroviral inhibitors.