Elongation activity of poliovirus RNA polymerase derived from Sabin type 1 sequence is not temperature sensitive.
Determinants of attenuation in the Sabin type 1 strain of poliovirus are located in the 5' noncoding region, the capsid coding region and the viral RNA-dependent RNA polymerase (3Dpol) coding region. These mutations also contribute to a temperature sensitive pheno-type of virus replication. We have cloned and expressed the Sabin 1 virus 3Dpol) protein which contains three amino acid differences from the wild-type (Mahoney) sequence, as well as a wild- type polymerase containing only a single Sabin amino acid substitution at nt 6203. These enzymes have been examined and compared for temperature sensitive polymerase activity. Wild-type and mutated polymerases demonstrated identical specific activities at 30, 35 and 39 degrees C. All three showed the same kinetics of heat inactivation after pre-incubation at elevated temperatures. Thus the contribution of Sabin 3Dpol sequences to the inability of the virus to grow at elevated temperatures must lie in a function or activity of the enzyme other than RNA polymerization. A likely reaction is the initiation step of RNA chain synthesis.
Baker
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Department of Molecular Biology and Biochemistry, University of California, Irvine 92717, USA.
Richards
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OC
Ehrenfeld
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D003001:Cloning, Molecular; D005814:Genes, Viral / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D009154:Mutation; D017955:Poliovirus / Q000201:enzymology* / Q000254:growth & development; D011055:Poliovirus Vaccine, Oral / Q000235:genetics*; D011072:Poly U / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014613:Vaccines, Attenuated; D015678:Viral Structural Proteins / Q000235:genetics
D011055:Poliovirus Vaccine, Oral; D011994:Recombinant Proteins; D014613:Vaccines, Attenuated; D015678:Viral Structural Proteins; D011072:Poly U; D012324:RNA-Dependent RNA Polymerase