Isolation of temperature sensitive mutants of Autographa californica nuclear polyhedrosis virus: Phenotype characterization of baculovirus mutants defective in very late gene expression

Abstract

Temperature sensitive mutants of Autographa californica nuclear polyhedrosis virus were isolated following exposure of parental replicating virus to 5-bromo-2′-deoxyuridine. Two mutants designated ts317 and ts538 were characterized in detail. Both mutants synthesized viral DNA but were defective in infectious extracellular virus production at the nonpermissive temperature suggesting defects in late gene regulation. Pulse-labeling experiments indicated that a switch to late protein synthesis occurred but this synthesis was not maintained with either mutant since all protein synthesis was gradually inhibited by 20 hr postinfection at 33°. Western blots probed with antisera directed against either extracellular virus proteins or very late proteins showed a reduction in late structural proteins, a 10-fold reduction in polyhedrin, and an absence of p10 at the nonpermissive temperature. Northern blots of total intracellular RNA showed that specific late and very late transcripts were synthesized in greatly reduced amounts at the nonpermissive temperature suggesting the absence of very late proteins was a result of inhibition of virus-specific transcription. The genetic analyses indicate that the mutations lie in two closely linked genes, both important in regulating the induction of very late transcription and translation.