Mutation of lysine residues in the nucleotide binding segments of the poliovirus RNA-dependent RNA polymerase.
The poliovirus 3D RNA-dependent RNA polymerase contains two peptide segments previously shown to cross-link to nucleotide substrates via lysine residues. To determine which lysine residue(s) might be implicated in catalytic function, we engineered mutations to generate proteins with leucine residues substituted individually for each of the lysine residues in the NTP binding regions. These proteins were expressed in Escherichia coli and were examined for their abilities to bind nucleotides and to catalyze RNA chain elongation in vitro. Replacement of each lysine residue in the NTP binding segment located in the central portion of the 3D molecule (Lys-276, -278, or -283) with leucine produced no impairment of GTP binding or polymerase activity. Substitution of leucine for Lys-61 in the N-terminal portion of the protein, however, abolished the binding of protein to GTP-agarose and all detectable polymerase activity. A nearby lysine replacement with leucine at position 66 had no effect on enzyme activity. The three mutations in the central region of 3D were introduced into full-length viral cDNAs, and the infectivities of RNA transcripts were examined in transfected HeLa cells. Growth of virus containing 3D with a mutation at residue 278 (3Dmu278) or 3Dmu283 was indistinguishable from that of the wild type; however, 3Dmu276 generated extremely slow-growing, small-plaque virus. Polyprotein processing by 3CDmu276 was unaffected. Large-plaque variants, in which the Leu-276 codon had mutated again to an arginine codon, emerged at high frequency. The results suggest that a lysine residue at position 61 of 3Dpol is essential for polymerase catalytic function and that a basic (lysine or arginine) residue at position 276 is required for some other function of 3D important for virus growth but not for RNA chain elongation or polyprotein processing.
Richards
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OC
Department of Molecular Biology and Biochemistry, University of California, Irvine 92697, USA.
Baker
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S
Ehrenfeld
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D002847:Chromatography, Agarose; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008239:Lysine; D016296:Mutagenesis; D017955:Poliovirus / Q000201:enzymology*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D006160:Guanosine Triphosphate; D012324:RNA-Dependent RNA Polymerase; D008239:Lysine