Structural, biochemical and functional characterization of the essential host factor SETD3 in complex with the enterovirus CV-B3 2A protease

Enteroviruses cause a number of medically relevant and widespread human diseases with no approved antiviral therapies currently available. We have previously identified the actin histidine methyltransferase SETD3 as critically important for enterovirus infection through a protein-protein interaction with the viral protease 2A. Here, we report the cryo-EM structure of SETD3 in complex with coxsackievirus B3 2A at 3.5 Å resolution. 2A interacts with two distinct interfaces on SETD3, one in the SET domain that overlaps with the actin binding interface, and one in the RSB domain. Structure-function analysis revealed that mutations of key residues in the SET interaction interface resulted in severely reduced binding to 2A and a complete protection from enteroviral infection. Altogether, our findings provide key structural insights into the interaction between SETD3 and 2A and provide a framework for the rational design of host targeted therapeutics against this class of viruses.