Effects of P2 cleavage site mutations on poliovirus polyprotein processing

The poliovirus genome comprises a single open reading frame which is translated to give one large polyprotein. The proteolytic cascade involved in the processing of this polyprotein is not yet understood in full detail,particularly concerning the processing of P2-P3, the precursor to the viral nonstructural polypeptides, 2A, 2B, 2C, 3A, 3B, 3C, and 3D. To investigate the possibility that the cleavage events within P2 and at the 2C/3A junction occur in an ordered fashion, we used oligonucleotide-directed mutagenesis of poliovirus cDNA to modify the 3C(prn)-mediated cleavage sites. The Gin residue of the Gin-Gly sequence at the 2A/2B, 2B/2C, and 2C/3A junctions in the poliovirus polyprotein was replaced by Asn, Glu, Asp, or Lys. The effects of each of these substitutions were studied in vivo after transfection onto HeLa cells and in vitro in a cell-free translation assay, using full-length mutated RNA transcripts. Only the mutant with the Glu-Gly sequence at the 2C/3A junction was viable. Analysis of the in vitro processing profiles showed that the efficiency of the 3C protease cleavage at any of the sites in P2 was in the following order: Gin-Gly > Glu-Gly > Asn-Gly. No cleavage could be detected with the Asp-Gly or Lys-Gly sequence at any junction. Lack of 2A/2B or 2B/2C cleavage had no consequences on the cleavage efficiency at other Gin-Gly sites in the polyprotein. Abolition of cleavage at the 2C/3A junction did not prevent the generation of the 2A, 2B, and 3CD polypeptides. Thus, these polypeptides. Thus, these polypeptides could be produced independently of the generation of the P2 and P3 precursors.