Comparison of Identical Temperature-Sensitive Mutations in the L Polymerase Proteins of Sendai and Parainfluenza3 Viruses

AbstractThe L subunit of the RNA-dependent RNA polymerase of negative strand RNA viruses is believed to possess all the enzymatic activities necessary for viral transcription and replication. Mutations in the L proteins of human parainfluenza virus type 3 (PIV3) and vesicular stomatitis virus (VSV) have been shown to confer temperature sensitivity to the viruses; however, their specific defects have not been determined. Mutant PIV3 L proteins expressed from plasmids were tested for temperature sensitivity in transcription and replication in a minigenome reporter system in cells and for in vitro transcription from purified PIV3 template. The single L mutants, Y942H and L992F, were temperature sensitive (ts) in both assays, although viral RNA synthesis was not completely abolished at the nonpermissive temperature. Surprisingly, the T1558I L mutant was not ts, although its cognate virus was ts. Thus the ts defect in this virus may be due to the abrogation of an essential interaction of the mutant polymerase with a host cell component, which is not measured by the RNA synthesis assays. Most of the combinations of the PIV3 L mutations were not additive and did not show temperature sensitivity in in vitro transcription. Since they were ts in the minigenome assay in vivo, replication appears to be specifically defective. The ts mutations in PIV3 and VSV L proteins were also substituted into the Sendai L protein to compare the defects in related systems. Only Sendai Y942H L was ts in both transcription and replication. One Sendai L mutant, L992F, gave much better replication than transcription. Several other mutants could transcribe but not replicate in vitro, while replication in vivo was normal