Expression and partial purification of recombinant tomato ringspot nepovirus 3C-like proteinase: comparison of the activity of the mature proteinase and the VPg-proteinase precursor

The 3C-like proteinase (Pro) from Tomato ringspot virus (genus Nepovirus) i s responsible for the processing of the RNA1-encoded (PI) and RNA2-encoded (P2) polyproteins. Cleavage between the VPg and Pro domains is inefficient in vitro and in E. coli, resulting in the accumulation of the VPg-Pro. In t his study, we have compared the trans-activity of the Pro and VPg-Pro on va rious P1- and P2-derived precursors. Recombinant Pro and VPg-Pro were parti ally purified using an E. coli expression system. A mutation of the VPg-Pro cleavage site was introduced into the VPg-Pro to prevent slow release of t he Pro. The Pro was five to ten times more active than the VPg-Pro on two P 2 cleavage sites (at the N- and C-termini of the movement protein domain) a nd was approximately two times more active than the VPg-Pro on the third P2 cleavage site (between the X3 and X4 domains). Neither the Pro nor the VPg -Pro could cleave in trans PI-derived substrates containing the three cleav age sites delineating the X1, X2, putative NTP-binding protein and VPg doma ins. These results are discussed in light of the possible regulation of the proteinase activity during virus replication. Crown Copyright (C) 2001 Els evier Science B.V. All rights reserved.