Determination of the mutation rate of poliovirus RNA-dependent RNA polymerase

The fidelity of poliovirus RNA-dependent RNA polymerase (3Dpol) was determined using a system based on the fidelity of synthesis of the α-lac gene which codes for a subunit of β-galactosidase. Synthesis products are screened for mutations by an α-complementation assay, in which the protein product from α-lac is used in trans to complement β-galactosidase activity in bacteria that do not express α-Lac. Several polymerases have been analyzed by this approach allowing comparisons to be drawn. The assay included RNA synthesis by 3Dpol on an RNA template that coded for the N-terminal region of α-Lac. 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 MMLV-RT. 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 α-lac. Results showed a mutation rate for 3Dpol corresponding to ≈4.5×10−4 errors per base (one error in ≈2200 bases). Analysis of mutations showed that base substitutions occurred with greater frequency than deletions and insertions.