EFFECTS OF P2 CLEAVAGE SITE MUTATIONS ON POLIOVIRUS POLYPROTEIN PROCESSING

The poliovirus genome comprises a single open reading frame which is t ranslated to give one large polyprotein. The proteolytic cascade invol ved in the processing of this polyprotein is not yet understood in ful l detail, particularly concerning the processing of P2-P3, the precurs or to the viral nonstructural polypeptides, 2A, 2B, 2C, 3A, 3B, 3C, an d 3D. To investigate the possibility that the cleavage events within P 2 and at the 2C/3A junction occur in an ordered fashion, we used oligo nucleotide-directed mutagenesis of poliovirus cDNA to modify the 3C(pr o)-mediated cleavage sites. The Gin residue of the Gln-Gly sequence at the 2A/2B, 2B/2C, and 2C/3A junctions in the poliovirus polyprotein w as replaced by Asn, Glu, Asp, or Lys. The effects of each of these sub stitutions 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/3 A junction was viable. Analysis of the in vitro processing profiles sh owed that the efficiency of the 3C protease cleavage at any of the sit es in P2 was in the following order: Gln-Gly >Glu-Gly >Asn-Gly. No cle avage could be detected with the Asp-Gly or Lys-Gly sequence at any ju nction. Lack of 2A/2B or 2B/2C cleavage had no consequences on the cle avage efficiency at other Gln-Gly sites in the polyprotein. Abolition of cleavage at the 2C/3A junction did not prevent the generation of th e 2A, 2B, and 3CD polypeptides. Thus, these polypeptides could be prod uced independently of the generation of the P2 and P3 precursors. (C) 1996 Academic Press. Inc.