STUDIES ON RNA POLYMERASE OF SOME TEMPERATURE-SENSITIVE MUTANTS OF SEMLIKI FOREST VIRUS

The polymerase which catalyzes the synthesis of the double-stranded form of viral RNA has been isolated from BHK21 cells infected with a number of temperature-sensitive mutants of Semliki forest virus, and their properties investigated. The development of RNA polymerase activity in cells infected with two mutants which made normal (ts 28) and subnormal (ts 15) amounts of viral RNA was similar to that of wild type at both the permissive (28°) and nonpermissive (38.5°) temperatures. The properties of the isolated enzymes did not differ significantly from those of the wild-type polymerase. Three mutants which synthesized no viral RNA at the nonpermissive temperature did not develop any RNA polymerase activity at this temperature. Polymerase prepared from cells infected at 28° with two of these mutants (ts 3 and ts 6) did not exhibit any marked thermal instability (compared with wild type) when incubated at various temperatures in vitro. Once formed at the lower temperature, the polymerase activities of cells infected with ts 3 and ts 6 were stable to further incubation at the nonpermissive temperature. The polymerase of the third mutant (ts 5) was slightly more unstable at elevated temperatures than the wild-type polymerase, but the difference was hardly sufficient to explain the temperature sensitivity of virus growth on the basis of a thermolabile polymerase. It was concluded that the synthesis or assembly of the ts 3 and ts 6 polymerases was sensitive to temperature, rather than the enzyme itself. The implications of this observation are discussed in terms of the temperature sensitivity of viral protein synthesis in general. © 1969.