Construction of a "mutagenesis cartridge" for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants.

Abstract

By following a strategy of genetic analysis of poliovirus, we have constructed a synthetic "mutagenesis cartridge" spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type 1 (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed us to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alteration of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. We suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).