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Temperature Sensitivity of the Transcriptase of Mutants tsB1 and tsF1 of Vesicular Stomatitis Virus New Jersey Is a Consequence of Mutation Affecting Polypeptide L

J. Ongrádi

Medical Research Council Virology Unit, Institute of Virology, University of Glasgow, Glasgow G11 5JR, U.K.

Two conditional transcriptase-negative mutants of vesicular stomatitis virus (VSV) serotype New Jersey, tsB1 and tsF1, their revertants tsB1/R1 and tsF1/R1 and the wild-type virus were dissociated into pellet, NS and L fractions and, after reconstitution of these in various combinations, the transcriptase activities were assayed in vitro at the permissive (31 °C) and restrictive (39 °C) temperatures. The pellet fractions contained the virion RNA-polypeptide N complexes, while the NS and L fractions were essentially pure preparations of these polypeptides. The synthesis of RNA by the reconstituted pellet and L fractions was inhibited at 39 °C only when the L fractions of tsB1 or tsF1 were used. Addition of the NS fractions to the reconstituted pellet and L fractions did not alter the rates of RNA synthesis. These results demonstrate that polypeptide L is the temperature-sensitive polypeptide of both mutants tsB1 and tsF1 and support previous observations that polypeptide L is the transcriptase itself. The fact that a second mutant of complementation group F, tsF2, is transcriptase-positive but replicase-negative suggests that polypeptide L is involved both in transcription and replication. Intracistronic complementations may account for the observation that the temperature-sensitive mutations affect polypeptide L in complementation groups B and F.

Keywords: VSV, tsB1 and tsF1, dissociation-reconstitution, transcription in vitro

Present address: Department of Microbiology, Semmelweis University of Medical Sciences, Budapest 1089, Hungary.

Received 11 March 1985; accepted 19 March 1985.