EFFICIENT AUTOPROTEOLYTIC PROCESSING OF THE MHV-A59 3C-LIKE PROTEINASE FROM THE FLANKING HYDROPHOBIC DOMAINS REQUIRES MEMBRANES

The replicase gene of the coronavirus MHV-A59 encodes a serine-like pr oteinase similar to the 3C proteinases of picornaviruses. This protein ase domain is flanked on both sides by hydrophobic, potentially membra ne-spanning, regions. Cell-free expression of a plasmid encoding only the 3C-like proteinase (3CLpro) resulted in the synthesis of a 29-kDa protein that was specifically recognized by an antibody directed again st the carboxy-terminal region of the proteinase. A protein of identic al mobility was detected in MHV-A59-infected cell lysates. In vitro ex pression of a plasmid encoding the 3CLpro and portions of the two flan king hydrophobic regions resulted in inefficient processing of the 29- kDa protein. However, the efficiency of this processing event was enha nced by the addition of canine pancreatic microsomes to the translatio n reaction, or removal of one of the flanking hydrophobic domains. Pro teolysis was inhibited in the presence of N-ethylmaleimide (NEM) or by mutagenesis of the catalytic cysteine residue of the proteinase, indi cating that the 3CLpro is responsible for its autoproteolytic cleavage from the flanking domains. Microsomal membranes were unable to enhanc e the trans processing of a precursor containing the inactive proteina se domain and both hydrophobic regions by a recombinant 3CLpro express ed from Escherichia coil. Membrane association assays demonstrated tha t the 29-kDa 3CLpro was present in the soluble fraction of the reticul ocyte lysates, while polypeptides containing the hydrophobic domains a ssociated with the membrane pellets. With the help of a viral epitope tag, we identified a 22-kDa membrane-associated polypeptide as the pro teolytic product containing the amino-terminal hydrophobic domain. (C) 1997 Academic Press.