Cleavage activity of the sapovirus 3C-like protease in Escherichia coli.
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.
Oka
T
T
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan. oka-t@nih.go.jp
Katayama
K
K
Ogawa
S
S
Hansman
G S
GS
Kageyama
T
T
Miyamura
T
T
Takeda
N
N
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007223:Infant; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D029341:Sapovirus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D010450:Endopeptidases