Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete...

All proteins encoded by the plant potyvirus, tobacco etch virus (TEV), arise by proteolytic processing of a single polyprotein. Two virus-encoded proteinases (NIa and HC-Pro) that catalyze most of the proteolytic events have been characterized previously. The two proteins that are derived from the N-terminal 87 kd region of the viral polyprotein are a 35 kd protein and HC-Pro (52 kd). It is demonstrated in this study that a third proteolytic activity is required to process the junction between these proteins. Proteolysis at the HC-Pro N terminus to separate these proteins occurred poorly, if at all, after in vitro synthesis of a 97 kd polyprotein, whereas cleavage of the HC-Pro C terminus occurred efficiently by an autoprocessing mechanism. Synthesis of the same polyprotein in transgenic tobacco plants, however, resulted in complete and accurate proteolysis at both termini of HC-Pro. A point mutation affecting an amino acid residue essential for the proteolytic activity of HC-Pro had no effect on N-terminal processing. Expression in transgenic plants of a construct with a large deletion in the 35 kd protein coding region resulted in partial inhibition of HC-Pro N-terminal cleavage, suggesting that the 35 kd protein may affect the proteolytic event but not in a catalytic role. We speculate that this cleavage event is catalyzed by either a cryptic potyviral proteinase that requires a host factor or subcellular environment for activation, or possibly a host proteinase.