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

Virus Res. 2001 Nov 5;79(1-2):153-64. doi: 10.1016/s0168-1702(01)00344-6.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3C Viral Proteases
  • Binding Sites
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / isolation & purification
  • Cysteine Endopeptidases / metabolism*
  • Enzyme Precursors / genetics
  • Enzyme Precursors / isolation & purification
  • Enzyme Precursors / metabolism*
  • Gene Expression
  • Nepovirus / enzymology*
  • Nepovirus / genetics
  • Polyproteins / metabolism
  • RNA, Viral / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Substrate Specificity
  • Viral Core Proteins / genetics
  • Viral Core Proteins / isolation & purification
  • Viral Core Proteins / metabolism*
  • Viral Proteins / genetics
  • Viral Proteins / isolation & purification
  • Viral Proteins / metabolism*

Substances

  • Enzyme Precursors
  • Polyproteins
  • RNA, Viral
  • Recombinant Fusion Proteins
  • Viral Core Proteins
  • Viral Proteins
  • Cysteine Endopeptidases
  • 3C Viral Proteases