THE ENCEPHALOMYOCARDITIS VIRUS 3C PROTEASE IS A SUBSTRATE FOR THE UBIQUITIN-MEDIATED PROTEOLYTIC SYSTEM

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.