Poliovirus proteinase 3C: large-scale expression, purification, and specific cleavage activity on natural and synthetic substrates in vitro.
Proteinase 3C of poliovirus type 2 (Sabin) was expressed at 4% total protein in Escherichia coli. The protein was soluble and could be purified by a simple scheme. It was weakly active on the capsid precursor P1 (expressed in vitro), which contains two cleavage sites. The products of processing P1 were 1ABC and 1D (VP1). The activity was insensitive to Triton X-100. Crude extracts of cells infected with poliovirus type 1 (Mahoney) gave strong processing and yielded 1AB (VP0), 1C (VP3), and 1D in the same assay system but were sensitive to detergent. 3C from cell extracts that was separated from its precursors resembled the recombinant proteinase in its activity. Recombinant 3C cleaved the peptide dansyl-Glu-Glu-Glu-Ala-Met-Glu-Gln-Gly-Ile-Thr-Asn-Lys-NH2 at the Gln-Gly bond. We conclude that 3C is merely the core of the Gln-Gly-cleaving activity which processes P1 in vivo and that there is probably a hydrophobic contact between a larger 3C precursor and its P1 substrate which allows the second processing reaction: 1ABC, 1D----1AB, 1C, 1D.
Nicklin
M J
MJ
Department of Microbiology, State University of New York, Stony Brook 11794-8621.
Harris
K S
KS
Pallai
P V
PV
Wimmer
E
E
D000086042:3C Viral Proteases; D002213:Capsid / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000378:metabolism; D010957:Plasmids; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins
D004792:Enzyme Precursors; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases