Specificity of the polioviral proteinase 3C towards genetically engineered cleavage sites in the viral capsid.
In a study of the cleavage specificity of poliovirus proteinase 3Cpro, two mutant polioviruses were constructed to include putative 3Cpro cleavage sites in the BC loop of VP1. The BC loop of VP1 in the wild-type virus is the neutralization antigenic site IA, consisting of a continuous chain of nine amino acids (ASTTNKDKL). The first mutant, W1-1D-BC1, has four altered amino acids in the BC loop (ASTQGPGKL); the second mutant, W1-1D-BC2, has an insertion of nine amino acids in the BC loop (ASTGTAKVQGPGNKDKL). W1-1D-BC1 and W1-1D-BC2 were viable, grew to high titre and produced plaques of normal size. W1-1D-BC1 virions were resistant to proteolytic cleavage of the BC loop in vivo as well as upon incubation with a large excess of 3Cpro in vitro, although a synthetic decapeptide (PASTQGPGKL) containing the amino acids of the BC loop in W1-1D-BC1 was cleaved by 3Cpro. In contrast, W1-1D-BC2 yielded virus the VP1 of which was cleaved partially in vivo and completely when incubated with 3Cpro in vitro. Our results showed that an insertion of nine amino acids into the antigenic loop of poliovirus, representing a synthetic 3Cpro cleavage site, renders the loop susceptible to cleavage by proteinase 3Cpro, but that this cleavage is restricted if the loop is the length of that in the native virion. This result implies that, in this case, structural restrictions override sequence determinants for cleavage of the BC loop by 3Cpro.
Mirzayan
C
C
Department of Microbiology, State University of New York, Stony Brook 11794.
Ingraham
R
R
Wimmer
E
E
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D002213:Capsid / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005818:Genetic Engineering; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D017955:Poliovirus / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins
D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases