A poliovirus mutant defective for self-cleavage at the COOH-terminus of the 3C protease exhibits secondary processing defects.
By in vitro recombination between the wild-type full-length infectious cDNA of poliovirus and a clone generated by the construction of a cDNA bank from a chemically derived temperature-sensitive plurimutant, we obtained a mutant cDNA with a T to C change at nucleotide 5658. This mutation replaces the isoleucine at residue 74 of the viral protease 3C by a threonine. The mutant virus recovered after transfection exhibited a small-plaque phenotype, and was deficient for viral RNA synthesis. Both these defects were more marked at 39 than at 37 degrees. The mutation was introduced into a bacterial plasmid which expresses the 3C protease along with its flanking autocatalytic cleavage sites. Analysis of the cleavage products expressed in Escherichia coli provided direct evidence that the modification impaired cleavage at the COOH-terminus of 3C. Cleavage at this same site was partially defective in mutant virus-infected HeLa cells, reducing the production of mature 3C and the viral replicase, 3D. Cleavage of P1, the precursor to the capsid polypeptides, was apparently unaffected by this defect, whereas cleavage events within the P2 region of the genome occurred inefficiently. This is indicative of differential strategies for 3C-specific cleavage events in vivo.
Kean
K M
KM
Unité de Virologie Moléculaire, UA CNRS 545, Institut Pasteur, Paris, France.
Agut
H
H
Fichot
O
O
Wimmer
E
E
Girard
M
M
D004247:DNA / Q000235:genetics; D003673:Defective Viruses / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D017955:Poliovirus / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012367:RNA, Viral / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D004247:DNA; D010447:Peptide Hydrolases