Cleavage activity of the sapovirus 3C-like protease in Escherichia coli

Arch Virol. 2005 Dec;150(12):2539-48. doi: 10.1007/s00705-005-0591-0. Epub 2005 Aug 1.


We recently determined the ORF1 cleavage map of Mc10, a human sapovirus (SaV) strain, as follows: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. This cleavage was dependent on the viral encoded 3C-like protease. To identify the cleavage site of SaV ORF1, putative p70 (Pro-Pol) and p14-p70 (VPg-Pro-Pol) were expressed as N-terminal GST and C-terminal 6 x His-tag fusion proteins in Escherichia coli, and the expressed products were analyzed by SDS-PAGE and Western blotting. Our results indicated that the efficient proteolytic cleavage occurred between p14 (VPg) and p70 (Pro-Pol), and N-terminal amino acid sequencing revealed that the cleavage site was between E(1055) and A(1056). In contrast, the p70 (Pro-Pol) was not further cleaved. We also found that SaV protease cleaved the Q/G site within the rhinovirus 3C protease recognition site. Site-directed mutagenesis in a conserved GDCG motif of the protease completely abolished these proteolytic activities. This is the first report to identify the cleavage site of the SaV ORF1 polyprotein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Blotting, Western
  • Electrophoresis, Polyacrylamide Gel
  • Endopeptidases / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Humans
  • Infant
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Sapovirus / enzymology
  • Sapovirus / genetics
  • Sapovirus / metabolism*
  • Sequence Analysis, Protein
  • Substrate Specificity
  • Viral Proteins / genetics
  • Viral Proteins / isolation & purification
  • Viral Proteins / metabolism*


  • Recombinant Fusion Proteins
  • Viral Proteins
  • Endopeptidases