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

The replication of coronaviruses involves proteo-lytic processing of the gene 1 translation products, pp1a and pp1ab. One of the key enzymes in this process is predicted to be a virus-encoded 3C-like proteinase. In this report, we describe a bacterial system that has allowed us to express and charac-terize a recombinant murine coronavirus (MHV-JHM) 3C-like proteinase. The partially purified protein has been shown to exhibit proteolytic activity in trans and mutation analysis has been used to demonstrate the indispensability of Cys-3495 for enzymatic activity. Finally, the effect of class-specific proteinase inhibitors on the trans cleavage activity of the MHV 3C-like proteinase has been used to demonstrate the functional and structural homology of this enzyme to the picorna-virus 3C proteinases. The coronavirus gene 1 encompasses approximately 20–22 kb and is composed of two open reading frames (ORFs)