Effect of reaction conditions and 3AB on the mutation rate of poliovirus RNA-dependent RNA polymerase in a α-complementation assay

The fidelity of poliovirus RNA-dependent RNA polymerase (3Dpol) was determined using a modified α-complementation assay. Several polymerases have been analyzed by this approach allowing comparisons to be drawn. Various conditions including high and low MgCl2, replacing MgCl2 with MnCl2, skewed nucleotide pools, and the presence of poliovirus protein 3AB were analyzed. The assay included RNA synthesis by 3Dpol on an RNA template that coded for a region of the alpha peptide of β-galactosidase (lacZ-α). The product of this reaction was used as a template for a second round of 3Dpol synthesis and the resulting RNA was reverse transcribed to DNA by reverse transcriptase. The DNA was amplified by PCR and inserted into a vector used to transform Escherichia coli. The bacteria were screened for β-galactosidase activity by blue–white phenotype analysis with white or faint blue colonies scored as errors made during synthesis on lacZ-α. Although 3AB strongly stimulated 3Dpol synthesis as expected, no change in fidelity was detected. Changes in MgCl2 also showed little effect. Mutation rates of ∼9 × 10−5 (∼1 error per 11,000 incorporations) were estimated for these conditions. In contrast, MnCl2 or skewed nucleotide pools were highly mutagenic resulting in lowered fidelity.