Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing.
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.
Carrington
J C
JC
Department of Biology, Texas A&M University, College Station 77843.
Freed
D D
DD
Oh
C S
CS
D000906:Antibodies; D015151:Immunoblotting; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D010942:Plant Viruses / Q000235:genetics*; D010947:Plants, Toxic; D011499:Protein Processing, Post-Translational; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D014764:Viral Proteins; D010447:Peptide Hydrolases