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) is responsible for the processing of the RNA1-encoded (P1) 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 this study, we have compared the trans-activity of the Pro and VPg-Pro on various P1- and P2-derived precursors. Recombinant Pro and VPg-Pro were partially 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 the Pro. The Pro was five to ten times more active than the VPg-Pro on two P2 cleavage sites (at the N- and C-termini of the movement protein domain) and 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 P1-derived substrates containing the three cleavage sites delineating the X1, X2, putative NTP-binding protein and VPg domains. These results are discussed in light of the possible regulation of the proteinase activity during virus replication.
Chisholm
J
J
Pacific Agri-Food Research Centre, BC, V0H 1Z0, Summerland, Canada.
Wieczorek
A
A
Sanfaçon
H
H
D000086042:3C Viral Proteases; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D017860:Nepovirus / Q000201:enzymology* / Q000235:genetics; D020815:Polyproteins / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014758:Viral Core Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004792:Enzyme Precursors; D020815:Polyproteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases