In vitro transcription of herpes simplex virus genes: identification of a new initiation site and second intervening sequence in the immediate-early RNA-5 gene.

We have used partially purified preparations of RNA polymerase II from mock-infected and herpes simplex virus type 1-infected HEp-2 cells to transcribe the herpes simplex virus type 1 strain F BamHI Z DNA fragment containing the promoter for immediate-early RNA-5 (alpha 47), a 1.8-kilobase, spliced RNA. In agreement with the in vivo transcriptional regulation of herpes simplex virus type 1 immediate-early genes, electrophoretic analyses of runoff and truncated transcripts from this template showed that RNA polymerase II from mock-infected cells initiates transcription more selectively than does that from the herpes virus-infected cells at the immediate-early RNA-5 promoter. S1 nuclease mapping of the 5' ends of in vivo- and in vitro-synthesized mRNA placed the initiation site ca. 110 base pairs upstream from the previously published site and also demonstrated the presence of a second, smaller intervening sequence between this new cap site and the previously characterized intervening sequence. S1 analyses also suggested that some splicing of the larger but not the smaller intervening sequence occurred in vitro.