title: Site-specific mutations at a picornavirus VP3/VP1 cleavage site disrupt in vitro processing and assembly of capsid precursors.
abstract: Most proteolytic cleavages within the picornavirus polyproteins are carried out by viral protease 3C. For encephalomyocarditis virus, the protease 3C-catalyzed processing occurs between Gln-Gly or Gln-Ser amino acid pairs which are flanked by proline residues, but the sequence-specific constraints on recognition and cleavage by the enzyme are not completely understood. To examine alternative cleavage site sequences, we constructed a cDNA plasmid which expresses the viral L-P1-2A capsid precursor in vitro and introduced site-specific mutations into the Gln-Gly pair at the VP3/VP1 junction. The altered protein substrates were tested for cleavage activity in assays with protease 3C. The encephalomyocarditis virus 3C processed Gln-Ala as efficiently as its natural sites but did not cleave Gln-Val, Gln-Glu, Lys-Gly, Lys-Ala, Lys-Val, Lys-Glu, or Pro-Gly combinations. Displacement of the flanking proline residue by an engineered insertion slowed but did not prevent cleavage at this site. Also, a mutant defective in processing at the VP3/VP1 junction was unable to form 14S pentameric assembly intermediates in vitro.
authors.affiliation: Institute for Molecular Virology, University of Wisconsin, Madison 53706.
authors.forename: G D
authors.initials: GD
authors.lastname: Parks
authors.forename: A C
authors.initials: AC
authors.lastname: Palmenberg
mesh_terms: D001483:Base Sequence; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004686:Encephalomyocarditis virus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D007118:Immunoassay; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014158:Transcription, Genetic
chemical_list: D004279:DNA, Viral; D011498:Protein Precursors; D010447:Peptide Hydrolases