MUTATION OF LYSINE RESIDUES IN THE NUCLEOTIDE-BINDING SEGMENTS OF THEPOLIOVIRUS RNA-DEPENDENT RNA-POLYMERASE

The poliovirus 3D RNA-dependent RNA polymerase contains two peptide se gments previously shown to cross-link to nucleotide substrates via lys ine residues, To determine which lysine residue(s) might be implicated in catalytic function, we engineered mutations to generate proteins w ith leucine residues substituted individually for each of the lysine r esidues in the NTP binding regions, These proteins were expressed in E scherichia coli and mere examined for their abilities to bind nucleoti des and to catalyze RNA chain elongation in vitro, Replacement of each lysine residue in the NTP binding segment located in the central port ion of the 3D molecule (Lys-276, -278, or -283) with leucine produced no impairment of GTP binding or polymerase activity, Substitution of l eucine for Lys-61 in the N-terminal portion of the protein, however, a bolished the binding of protein to GTP-agarose and all detectable poly merase activity, A nearby lysine replacement with leucine al: position 66 had no effect on enzyme activity, The three mutations in the centr al region of 3D were introduced into full-length viral cDNAs, and the infectivities of RNA transcripts were examined in transfected HeLa cel ls. Growth of virus containing 3D with a mutation at residue 278 (3D m u 278) or 3D mu 283 was indistinguishable from that of the wild type; however 3D mu 276 generated extremely slow-growing, small-plaque virus . Polyprotein processing by 3CD mu 276 was unaffected. Large-plaque va riants, in which the Leu-276 codon had mutated again to an arginine co don, emerged at high frequency, The results suggest that: a lysine res idue at position 61 of 3D(pol) is essential for polymerase catalytic f unction and that a basic (lysine or arginine) residue at position 276 is required for some other function of 3D important for virus growth b ut not for RNA chain elongation or polyprotein processing.