Temperature-Sensitive Mutations in the Protein Kinase-1 (pk-1) Gene of theAutographa californicaNuclear Polyhedrosis Virus That Block Very Late Gene Expression

Abstract

We have developed a genetic screen for temperature-sensitive mutations in the very late transcription apparatus of theAutographa californicanuclear polyhedrosis virus. This method starts with the BacPAK6 virus, which has theEscherichia coli lacZgene under the control of the very late polyhedrin promoter. The desired mutants are temperature-sensitive for β-galactosidase production and can be complemented by wild-type virus, which lacks thelacZgene. Two mutants created by nitrosoguanidine mutagenesis and identified by this screen, and one mutant identified by another screen, have been mapped by marker rescue to the viral protein kinase 1 gene (pk-1). The protein kinase genes of these three mutant viruses have been sequenced, revealing the same point mutation in two of them and a different point mutation in the other. In each case, a single amino acid is changed: In two mutants, XF4 and XF5, Asp 92 is changed to Asn; in the other mutant, KT800, Thr 204 is changed to Ile. Northern blotting of RNA made in cells infected by these three mutant viruses has shown that the accumulation of very late transcripts (lacZandp10) is temperature-sensitive, but that accumulation of at least one late transcript (vp39) is not temperature-sensitive. Nuclear run-on transcription assays with two of the mutants indicate that very late transcription is somewhat temperature-sensitive, although this defect is not as pronounced as the temperature-sensitivity detected by Northern blotting. Transcription of at least one late gene (vp39) is not temperature-sensitive in cells infected by these two mutants. Thus, it appears that the viral protein kinase-1 is involved in very late gene expression. Some of this effect is at the transcription level, but some may also be exerted at the posttranscription level.