Plasmid Construction for FMDV 3C Protease Assay

Plasmids carrying either a native 3ABCD ORF (p3ABCD) or a 3ABCD ORF containing mutated 3C^pro with Cys142Ser and Cys163Gly (pmu3ABCD) were generated as previously described [45 (link)] with slight modification. The primer sequences for PCR cloning are presented in Supplementary Materials Table S1. Plasmids pBIND-VP16 and pG5luc were purchased from Promega, Madison, WI, USA [46 (link)]. To produce pBIND_FMDV 3C^pro plasmids for use in the protease assay, the 3ABCD and mu3ABCD DNA fragments in p3ABCD and pmu3ABCD were subcloned into pBIND-VP16 plasmid (Promega, Madison, WI, USA) between a GAL4-binding domain and a VP16 activation domain at the BamH I and Mlu I sites, resulting in pBV_3ABCD and pBV_mu3ABCD, respectively. The pG5luc containing the GAL4-binding site upstream of the firefly luciferase gene was used as a reporter system while pBV_3ABCD and pBV_mu3ABCD also carried the Renilla luciferase gene, which served as an internal luciferase control.