Comparison of identical temperature-sensitive mutations in the L polymerase proteins of Sendai and parainfluenza3 viruses

The L subunit of the RNA-dependent RNA polymerase of negative strand RNA vi ruses is believed to possess all the enzymatic activities necessary for vir al transcription and replication. Mutations in the L proteins of human para influenza virus type 3 (PIV3) and vesicular stomatitis virus (VSV) have bee n shown to confer temperature sensitivity to the viruses; however, their sp ecific defects have not been determined. Mutant PIV3 L proteins expressed f rom plasmids were tested for temperature sensitivity in transcription and r eplication in a minigenome reporter system in cells and for in vitro transc ription from purified PIV3 template. The single L mutants, Y942H and L992F, were temperature sensitive (ts) in both assays, although viral RNA synthes is was not completely abolished at the nonpermissive temperature. Surprisin gly, the T1558I L mutant was not ts, although its cognate virus was ts. Thu s 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 n ot measured by the RNA synthesis assays. Most of the combinations of the PI V3 L mutations were not additive and did not show temperature sensitivity i n in vitro transcription. Since they were ts in the minigenome assay in viv o, replication appears to be specifically defective. The ts mutations in PI V3 and VSV L proteins were also substituted into the Sendai L protein to co mpare the defects in related systems. Only Sendai Y942H L was ts in both tr anscription and replication. One Sendai L mutant, L992F, gave much better r eplication than transcription. Several other mutants could transcribe but n ot replicate in vitro, while replication in vivo was normal. (C) 2000 Acade mic Press.