Clustered charged-to-alanine mutagenesis of poliovirus RNA-dependent RNA polymerase yields multiple temperature-sensitive mutants defective in RNA synthesis

Abstract

To generate a collection of conditionally defective poliovirus mutants, clustered charged-to-alanine mutagenesis of the RNA-dependent RNA polymerase 3D was performed. Clusters of charged residues in the polymerase coding region were replaced with alanines by deoxyoligonucleotide-directed mutagenesis of a full-length poliovirus cDNA clone. Following transfection of 27 mutagenized cDNA clones, 10 (37%) gave rise to viruses with temperature-sensitive (ts) phenotypes. Three of the ts mutants displayed severe ts plaque reduction phenotypes, producing at least 103-fold fewer plaques at 39.5°C than at 32.5°C; the other seven mutants displayed ts small-plaque phenotypes. Constant-temperature, single-cycle infections showed defects in virus yield or RNA accumulation at the nonpermissive temperature for eight stable ts mutants. In temperature shift experiments, seven of the ts mutants showed reduced accumulation of viral RNA at the nonpermissive temperature and showed no other ts defects. The mutations responsible for the phenotypes of most of these ts mutants lie in the N-terminal third of the 3D coding region, where no well-characterized mutations responsible for viable mutants had been previously identified. Clustered charged-to-alanine mutagenesis (S. H. Bass,

Highly similar structural frames link the template tunnel and NTP entry tunnel to the exterior surface in RNA-dependent RNA polymerases

“…(D) In PV, an insertion (red) at the C-terminal edge of the motif is lethal: L241-i-S242 (42). A species-specific loop (green) affects the catalytic rate (in PV) (37).…”

“…Each flank of the homomorph contains amino acid residues that significantly affect the life cycle of the species. In PV, mutations at the N-terminal residue of the homomorph (D71A/E72A) are lethal (37). Mutations located outside the N-terminal edge of the motif (PV D105A/E108A) result in small plaques (37).…”

“…In PV, mutations at the N-terminal residue of the homomorph (D71A/E72A) are lethal (37). Mutations located outside the N-terminal edge of the motif (PV D105A/E108A) result in small plaques (37). Downstream from the C-terminal edge of the motif, there is a nuclear localization signal (NLS) in the picornaviruses and caliciviruses.…”

“…Downstream from the C-terminal edge of the motif, there is a nuclear localization signal (NLS) in the picornaviruses and caliciviruses. The NLS is located two residues from the C-terminus of the homomorph and mutations in the NLS (K125A/ K126A/K127A and K127A/R128A/D129A) are lethal to PV (37).…”

“…Both the N-terminal and C-terminal residues of the homomorph are exposed at the exterior surface of the protein. In PV, mutations of residues adjacent to the N-terminal are lethal: G149-i-I150 (42) and H149A/K150A (37).…”