Expression and characterization of a recombinant murine coronavirus 3C- like proteinase

This study describes the bacterial expression and characterization of a recombinant 3C-like proteinase from murine coronavirus MHV-JHM, an enzyme central to coronavirus replication. The researchers expressed the proteinase as a fusion protein with maltose-binding protein in E. coli, then purified the active enzyme using factor Xa cleavage. Using a synthetic peptide substrate containing a known cleavage site, they demonstrated the enzyme's proteolytic activity and determined a turnover number of approximately 1.5 s⁻¹. Mutagenesis studies showed that the predicted catalytic residue Cys-3495 is essential for activity, as substitutions with glycine, alanine, valine, arginine, aspartate, or serine completely abolished proteolysis. The enzyme exhibited optimal activity between pH 7-8 and remained relatively insensitive to varying salt concentrations. Inhibitor studies revealed that the MHV 3C-like proteinase is inhibited by inhibitors targeting both serine and cysteine proteases, demonstrating functional and structural homology to picornavirus 3C proteinases. This bacterial expression system provides a practical approach for investigating coronavirus proteinase structure, catalytic mechanisms, and substrate specificity.

Key findings

• Recombinant MHV-JHM 3C-like proteinase was successfully expressed in E. coli and shown to have proteolytic activity in trans using a synthetic peptide substrate
• The predicted catalytic residue Cys-3495 is absolutely essential for enzymatic activity; all tested point mutations at this position eliminated proteolysis
• The enzyme exhibits a turnover number of ~1.5 s⁻¹, comparable to picornavirus 3C proteinases, with optimal activity at pH 7-8
• The MHV 3C-like proteinase shows inhibitor susceptibility patterns similar to picornavirus 3C proteinases, indicating structural and functional homology between coronavirus and picornavirus proteases