Autocatalytic Processing of the Potyvirus Helper Component Proteinase in Escherichia coli and In Vitro

The virus-encoded proteins of tobacco etch virus (TEV), a plant potyvirus, arise by proteolytic processing of\ud a large polyprotein precursor. The TEV genome codes for two proteinases, a 49-kilodalton proteinase and\ud helper component proteinase (HC-Pro), which cleave the polyprotein at specific sites. The only known cleavage\ud event catalyzed by HC-Pro occurs at the HC-Pro carboxyl terminus. The proteolytic activity of HC-Pro was\ud analyzed by expression of the enzyme in bacterial and cell-free systems. The carboxyl-terminal domain of\ud HC-Pro exhibited proteolytic activity in Escherichia coli with a processing half-time of approximately 100 s.\ud The processing kinetics of HC-Pro expressed in vitro by cell-free transcription and translation was variable,\ud depending on the presence or absence of TEV polypeptide sequences at the amino terminus of the proteolytic\ud domain. Cleavage of the HC-Pro carboxyl terminus appeared to proceed exclusively by an autocatalytic\ud mechanism; the proteinase synthesized in vitro exhibited little or no proteolytic activity when reacted with the\ud HC-Pro cleavage site in trans or bimolecular reactions