Temperature-sensitive polioviruses containing mutations in RNA polymerase.
Site-directed mutagenesis was performed to change the wild-type residue (asparagine) to aspartate, histidine, or tyrosine at amino acid 424 of the poliovirus RNA polymerase, 3Dpol. The mutations were introduced into plasmids containing full-length viral cDNA and plasmids which direct the expression of 3Dpol in Escherichia coli. Mutant viruses, recovered after transfection of HeLa cells with RNA transcripts of the full-length clones, produced small plaques at 32 degrees. In addition, the plaquing efficiency was decreased for all three mutants at 37 degrees, compared to 32 degrees. The polyprotein processing of all mutant viruses was normal at the temperatures tested, suggesting that the mutant plaque phenotypes were not due to incorrect processing of viral proteins. Analyses of viral RNA synthesis in infected cells and of the polymerase activities of mutant enzymes produced in E. coli suggested the following: (1) The his424 mutant enzyme appeared to be defective in the initiation of plus-strand RNA synthesis in HeLa cells. (2) The asp424 mutant enzyme appeared unable to assume proper conformation for active polymerase function when synthesized at 37 degrees. (3) The tyr424 mutant enzyme was totally inactive when synthesized in E. coli at 37 degrees.
Burns
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Department of Cellular, Viral and Molecular Biology, University of Utah School of Medicine, Salt Lake City 84132.
Richards
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Ehrenfeld
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D015153:Blotting, Western; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017955:Poliovirus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature; D010948:Viral Plaque Assay; D014779:Virus Replication
D011506:Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D012324:RNA-Dependent RNA Polymerase