Cleavage specificity of the poliovirus 3C protease is not restricted to Gln-Gly at the 3C/3D junction.
The 3C protease of poliovirus is distinguished from that of all other picornaviruses in that it only cleaves at Gln-Gly amino acid pairs within the viral polyprotein. To determine whether this strict cleavage specificity is an intrinsic property of the poliovirus 3C protease, amino acid substitutions were introduced at one of the Gln-Gly cleavage sites. Oligonucleotide-directed site-specific mutagenesis of an infectious poliovirus type 1 (Mahoney strain) cDNA was used to change the Gln-Gly site at the 3C/3D junction of the polyprotein into Gln-Val, Gln-Ala, Gln-Ser or Gln-Pro. The effects of these substitutions were studied in vivo after transfection of primate cells by the mutated cDNAs. The Gln-Gly to Gln-Pro substitution was lethal for virus growth, and the corresponding altered 3CD polypeptide expressed in insect cells using a recombinant baculovirus vector did not appear to undergo autocleavage. The Gln-Gly to Gln-Val change was also lethal, although production of virus was occasionally observed as a result of reverse mutations. Mutants with Gln-Ala and Gln-Ser sequences were viable, indicating that these dipeptides can be cleaved by the poliovirus protease in vivo. However, processing at the 3C/3D junction occurred relatively inefficiently in the case of the Gln-Ser virus. Furthermore, the Gln-Gly to Gln-Ala substitution seemed to result in an additional cleavage event within the N-terminal part of polypeptide 3D.
Kean
K M
KM
Unité de Virologie Moléculaire (CNRS UA 545), Institut Pasteur, Paris, France.
Teterina
N
N
Girard
M
M
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009036:Moths / Q000382:microbiology; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013379:Substrate Specificity; D014764:Viral Proteins
D004279:DNA, Viral; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases