Substitution mutations at the putative catalytic triad of the poliovirus 3C protease have differential effects on cleavage at different sites.
Picornavirus 3C proteases are substrate-specific cysteine proteases, proposed to be homologous to the trypsin/chymotrypsin-like serine proteases on the basis of structural predictions. Substitutions at the putative active-site residues (Glu71 and Cys147) of the poliovirus 3C protease did not completely abolish proteolytic processing in vitro. The activity of mutated 3C proteases was in the following hierarchy: Glu71-Cys147 (wild type) > Asp71-Cys147 > Glu71-Ser147 > Gln71-Cys147 > Asp71-Ser147 > Gln71-Ser147 (inactive at all sites). Such mutations had differential effects on cleavage at different sites of the poliovirus polyprotein. Cleavage within the P1 region of the polyprotein was the most defective, at the 1ABC/VP1 junction and particularly at the VP0/VP3 junction. Cleavage at the 3AB/3CD and 2B/2C junctions was less affected by the mutations, and the P2/P3 and 2A/2BC junctions were cleaved efficiently by all mutants except Gln71-Ser147. All the 3C mutants gave negative results in infectivity and replication assays after transfection, indicating that mutation of Glu71 or Cys147 virtually abolishes viral replication, irrespective of the efficiency of processing of the nonstructural part of the polyprotein.
Kean
K M
KM
Unité de Virologie Moléculaire (CNRS UA 545), Institut Pasteur, Paris, France.
Howell
M T
MT
Grünert
S
S
Girard
M
M
Jackson
R J
RJ
D000086042:3C Viral Proteases; D001665:Binding Sites; D002474:Cell-Free System; D003545:Cysteine / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D017354:Point Mutation; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D014162:Transfection; D014764:Viral Proteins; D014779:Virus Replication
D005971:Glutamates; D014764:Viral Proteins; D018698:Glutamic Acid; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases; D003545:Cysteine