Tg. Lawson et al., THE ENCEPHALOMYOCARDITIS VIRUS 3C PROTEASE IS A SUBSTRATE FOR THE UBIQUITIN-MEDIATED PROTEOLYTIC SYSTEM, The Journal of biological chemistry, 269(45), 1994, pp. 28429-28435
The encephalomyocarditis virus 3C protease has been shown to be rapidl
y degraded in infected cells and in vitro in rabbit reticulocyte lysat
e. The in vitro degradation, at least, is accomplished by a virus-inde
pendent, ATP-dependent proteolytic system. Here we identify this prote
olytic system as the ubiquitin-mediated system. Incubation of the 3C p
rotease in rabbit reticulocyte or cultured mouse cell lysate preparati
ons, alone or in the presence of added ubiquitin or methylated ubiquit
in, 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 u
biquitin. Supplemental ubiquitin also stimulated the degradation of th
e 3C protease in these preparations. Large 3C protease-polyubiquitin c
onjugates were observed to accumulate in reticulocyte lysate in the pr
esence of adenosine 5'-O-(3-thiotriphosphate), an inhibitor of the 26
S multicatalytic protease. This, combined with the fact that the prote
olytic activity could be removed from the lysate by sedimentation, imp
licates the multicatalytic protease in the degradation of the 3C prote
ase-ubiquitin conjugates. It was also found that the slow rate of degr
adation of a model polyprotein, which resembles the stable viral 3CD d
iprotein produced in vivo, is likely due to the fact that the polyprot
ein is a poor substrate for the ubiquitin conjugating system.