The encephalomyocarditis virus 3C protease is a substrate for the ubiquitin-mediated proteolytic system.
The encephalomyocarditis virus 3C protease has been shown to be rapidly degraded in infected cells and in vitro in rabbit reticulocyte lysate. The in vitro degradation, at least, is accomplished by a virus-independent, ATP-dependent proteolytic system. Here we identify this proteolytic system as the ubiquitin-mediated system. Incubation of the 3C protease in rabbit reticulocyte or cultured mouse cell lysate preparations, alone or in the presence of added ubiquitin or methylated ubiquitin, resulted in the generation of new higher molecular weight species. These new products were shown to be 3C protease-ubiquitin conjugates by their ability to bind antibodies against both the 3C protease and ubiquitin. Supplemental ubiquitin also stimulated the degradation of the 3C protease in these preparations. Large 3C protease-polyubiquitin conjugates were observed to accumulate in reticulocyte lysate in the presence of adenosine 5'-O-(3-thiotriphosphate), an inhibitor of the 26 S multicatalytic protease. This, combined with the fact that the proteolytic activity could be removed from the lysate by sedimentation, implicates the multicatalytic protease in the degradation of the 3C protease-ubiquitin conjugates. It was also found that the slow rate of degradation of a model polyprotein, which resembles the stable viral 3CD diprotein produced in vivo, is likely due to the fact that the polyprotein is a poor substrate for the ubiquitin-conjugating system.
Department of Chemistry, Bates College, Lewiston, Maine 04240.
T G
TG
Lawson
D L
DL
Gronros
J A
JA
Werner
A C
AC
Wey
A M
AM
DiGeorge
J L
JL
Lockhart
J W
JW
Wilson
P L
PL
Wintrode
D000086042:3C Viral Proteases; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004686:Encephalomyocarditis virus / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D014176:Protein Biosynthesis; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014452:Ubiquitins / Q000378:metabolism* / Q000494:pharmacology; D014764:Viral Proteins
D011994:Recombinant Proteins; D014452:Ubiquitins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases