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 proteinase similar to the 3C proteinases of picornaviruses. This proteinase domain is flanked on both sides by hydrophobic, potentially membrane-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 against the carboxy-terminal region of the proteinase. A protein of identical mobility was detected in MHV-A59-infected cell lysates. In vitro expression of a plasmid encoding the 3CLpro and portions of the two flanking hydrophobic regions resulted in inefficient processing of the 29-kDa protein. However, the efficiency of this processing event was enhanced by the addition of canine pancreatic microsomes to the translation reaction, or removal of one of the flanking hydrophobic domains. Proteolysis was inhibited in the presence of N-ethylmaleimide (NEM) or by mutagenesis of the catalytic cysteine residue of the proteinase, indicating that the 3CLpro is responsible for its autoproteolytic cleavage from the flanking domains. Microsomal membranes were unable to enhance the trans processing of a precursor containing the inactive proteinase domain and both hydrophobic regions by a recombinant 3CLpro expressed from Escherichia coli. Membrane association assays demonstrated that the 29-kDa 3CLpro was present in the soluble fraction of the reticulocyte lysates, while polypeptides containing the hydrophobic domains associated with the membrane pelletes. With the help of a viral epitope tag, we identified a 22-kDa membrane-associated polypeptide as the proteolytic product containing the amino-terminal hydrophobic domain.
Piñón
J D
JD
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6076, USA.
Mayreddy
R R
RR
Turner
J D
JD
Khan
F S
FS
Bonilla
P J
PJ
Weiss
S R
SR
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000821:virology*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000201:enzymology*; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases