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

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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 MgCl 2, replacing MgCl 2 with MnCl 2, 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 MgCl 2 also showed little effect. Mutation rates of ∼9 × 10 −5 (∼1 error per 11,000 incorporations) were estimated for these conditions. In contrast, MnCl 2 or skewed nucleotide pools were highly mutagenic resulting in lowered fidelity.