CLUSTERED CHARGED-TO-ALANINE MUTAGENESIS OF POLIOVIRUS RNA-DEPENDENT RNA-POLYMERASE YIELDS MULTIPLE TEMPERATURE-SENSITIVE MUTANTS DEFECTIVEIN RNA-SYNTHESIS

To generate a collection of conditionally defective poliovirus mutants , clustered charged-to-alanine mutagenesis of the RNA-dependent RNA po lymerase 3D was performed. Clusters of charged residues in the polymer ase 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 vir uses with temperature-sensitive (ts) phenotypes. Three of the ts mutan ts displayed severe ts plaque reduction phenotypes, producing at least 10(3)-fold fewer plaques at 39.5 degrees C than at 32.5 degrees C; th e other seven mutants displayed ts small-plaque phenotypes. Constant-t emperature, single-cycle infections showed defects in virus yield or R NA accumulation at the nonpermissive temperature for eight stable ts m utants. In temperature shift experiments, seven of the ts mutants show ed reduced accumulation of viral RNA at the nonpermissive temperature and showed no other ts defects. The mutations responsible for the phen otypes of most of these ts mutants lie in the N-terminal third of the 3D coding region, where no well-characterized mutations responsible fo r viable mutants had been previously identified. Clustered charged-to- alanine mutagenesis (S. Ii. Bass, M. G. Mulkerrin, and J. A. Wells, Pr oc. Natl. Acad. Sci. USA 88:4498-4502, 1991; W. F. Bennett, N. F. Paon i, B. A. Keyt, D. Botstein, J. J. S. Jones, L. Presta, F. M. Wurm, and M. J. ZoIler, J. Biol. Chem. 266:5191-5201, 1991; and K. F. Wertman, D. G. Drubin, and D. Botstein, Genetics 132:337-350, 1992) is designed to target residues on the surfaces of folded proteins; thus, extragen ic suppression analysis of such mutant viruses may be very useful in i dentifying components of the viral replication complex.