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

The poliovirus RNA-dependent RNA polymerase, 3D(Pol), is known to shar e a region of sequence homology with all RNA polymerases centered at t he GDD amino acid moth. The two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of t he enzyme. To test this hypothesis, we have utilized oligonucleotide s ite-directed mutagenesis to generate defined mutations in the aspartic acids of the GDD moth of the 3D(POl) gene. The codon for the first as partate (3D-D-328 [D refers to the single amino acid change, and the n umber refers to its position in the polymerase]) was changed to that f or 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 chang ed to that for glutamic acid or asparagine. The mutant enzymes were ex pressed in Escherichia coli, and the in vitro poly(U) polymerase activ ity was characterized. All of the mutant 3D(pol) enzymes were enzymati cally inactive in vitro when tested over a range of Mg2+ concentration s. 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 o f different transition metal ions were substituted for Mg2+ in the pol y(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(p ol) genes did not produce infectious virus when transfected into tissu e culture cells under standard conditions. Because of the activity of the 3D-N-329 mutant in the presence of Fe2+ and Mn2+, transfections we re also performed in the presence of the different metal ions. Surpris ingly, the transfection of the cDNA containing the 3D-N-329 mutation r esulted 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 i n CoCl2 grew at a rate which was similar to that of the wild-type poli ovirus. The nucleotide sequence of the virus obtained from transfectio n in the presence of Co2+ revealed that the 3D-N-329 mutation in the p olymerase had reverted to a 3D-D-329. These results demonstrate that a lthough the first aspartic acid residue is absolutely required for enz yme function, flexibility exists with respect to the requirement for t he second aspartic acid residue. The activity of the 3D-N-329 mutant i n the presence of different metal ions suggests the involvement of the aspartic acids in metal ion coordination during polymerization.