Abstract
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.
Highlights
The encephalomyocarditis viru3sC protease has been The 3C protease, produced either as a resuolftviral infection shown to be rapidly degraded in infected cells and in or from an inducible expression vector, has been found to be vitro in rabbit reticulocyte lysate
This, combined with the fact that the proteolytic activity could be removed the lysate by sedimentation, implicates the multicatalytic protease in the degradation of the 3C proteaseubiquitin conjugates
The successful replication of the virus requires that this polyprotein be processed by specific proteolytic cleavages into individual, functional proteins [1].All but two of these cleavages are catalyzed by a proteolytic activity which maps in the 3C region of the viral polyprotein [1, 2]
Summary
Plasmid Constructions and Preparation of RNAZkanscripts-The construction of thien vitro transcription plasmids pEBB’CD‘, pE3A’BCD, pE3B’CiD,and pE5LVPO has been described previously[5, 26]. pE3B’CD’ was modified by treatment with XhoI and Klenow fragment, followed by the insertion of the SpeI linker (New England Biolabs) d(CTAGACTAGTCTAG), which provides an in-frame stop codon. Terminated in vitro translation reaction mixtures (containing 58% nuclease-treated rabbit reticulocyte lysate with energy generating system, Promega)were incubated with or without added 0.1 m Ub (Sigma)at 30 O, and aliquots were removed a t various times for analysis by SDSPAGE, autoradiography and liquid scintillation counting ( 5 ) .The stability of the purified mature 3C protease in reticulocyte lysate and reticulocytesupernatant fraction was monitored by incubating 2 m 3C protease in the presence or absence of 0.2 mM added Ub or Me-Ub (prepared as described previously)(33)in reaction mixtures containing 20 nm HEPES-KOH,pH 7.8,lOO mM KC,H,O,, 1mM Mg(C,H,O,),, and 1mM DTT (buffer R) in 50%reticulocytelysate or supernatant fraction with an energy generating system [34].Ten-plaliquots were removedat various times and immunoprecipitated with optimal titers of anti-3C protease antibodies. Fifteen-pl aliquots were removed and immunoprecipitated with anti-3C protease antibodies, and the precipitated samples were analyzed by electrophoresis and Western blotting
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