MUTATION OF THE ASPARTIC-ACID RESIDUES OF THE GDD SEQUENCE MOTIF OF POLIOVIRUS RNA-DEPENDENT RNA-POLYMERASE RESULTS IN ENZYMES WITH ALTERED METAL-ION REQUIREMENTS FOR ACTIVITY

The poliovirus RNA-dependent RNA polymerase, 3D(Pol), is known to share a region of sequence homology with all RNA polymerases centered at the GDD amino acid moth. The two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of the enzyme. To test this hypothesis, we have utilized oligonucleotide site-directed mutagenesis to generate defined mutations in the aspartic acids of the GDD moth of the 3D(POl) gene. The codon for the first aspartate (3D-D-328 [D refers to the single amino acid change, and the number refers to its position in the polymerase]) was changed to that for glutamic acid, histidine, asparagine, or glutamine; the codons for both aspartic acids were simultaneously changed to those for glutamic acids; and the codon for the second aspartic acid (3D-D-329) was changed to that for glutamic acid or asparagine. The mutant enzymes were expressed in Escherichia coli, and the in vitro poly(U) polymerase activity was characterized. All of the mutant 3D(pol) enzymes were enzymatically inactive in vitro when tested over a range of Mg2+ concentrations. However, when Mn2+ was substituted for Mg2+ in the in vitro assays, the mutant that substituted the second aspartic acid for asparagine (3D-N-329) was active. To further substantiate this finding, a series of different transition metal ions were substituted for Mg2+ in the poly(U) polymerase assay. The wild-type enzyme was active with all metals except Ca2+, while the 3D-N-329 mutant was active only when FeC6H7O5 was used in the reaction. To determine the effects of the mutations on poliovirus replication, the mutant 3D(POl) genes were subcloned into an infectious cDNA of poliovirus. The cDNAs containing the mutant 3D(pol) genes did not produce infectious virus when transfected into tissue culture cells under standard conditions. Because of the activity of the 3D-N-329 mutant in the presence of Fe2+ and Mn2+, transfections were also performed in the presence of the different metal ions. Surprisingly, the transfection of the cDNA containing the 3D-N-329 mutation resulted in the production of virus at a low frequency in the presence of FeSO4 or CoCl2. The virus derived from transfection in the presence of FeSO4 grew slowly, while the viruses recovered from transfection in CoCl2 grew at a rate which was similar to that of the wild-type poliovirus. The nucleotide sequence of the virus obtained from transfection in the presence of Co2+ revealed that the 3D-N-329 mutation in the polymerase had reverted to a 3D-D-329. These results demonstrate that although the first aspartic acid residue is absolutely required for enzyme function, flexibility exists with respect to the requirement for the second aspartic acid residue. The activity of the 3D-N-329 mutant in the presence of different metal ions suggests the involvement of the aspartic acids in metal ion coordination during polymerization.