Regulation of herpes simplex virus gene transcription in vitro

We used partially purified RNA polymerase II from uninfected (Pol II) and from herpes simplex virus type 1 (HSV-1) infected HEp-2 cells (Pol II-H) to transcribe HSV-1 DNA in vitro. Gel electrophoretic analysis of the products produced from native HSV-1 DNA yielded weight average chain lengths of 4.0 and 3.5 kb for the Pol II and Pol II-H products, respectively. Blot hybridization analyses of the HSV DNA transcripts showed that both enzymes transcribed RNA from essentially all regions of the genome. However, Pol II preferentially transcribed regions coding for the immediate-early or alpha mRNAs, whereas Pol II-H preferentially copied regions coding for the early (beta) and late (gamma) gene products. Transcriptional analyses of the cloned HSV-1 Bam HI-Q fragment (containing the thymidine kinase (TK) gene) and its subfragments showed that (1) the major transcripts produced by Pol II-H were distinctly different from those produced by Pol II; (2) Pol II and Pol II-H utilized different promoters for the synthesis of major transcripts; (3) both enzymes produced three minor transcripts that were partially overlapping and in opposite direction to the TK gene; and (4) only Pol II-H initiated transcription from the TK promoter. In contrast, both Pol II and Pol II-H generated an identical set of transcripts from an adenovirus 2 early region DNA fragment. The sizes of the products suggest that RNA processing may be occurring in vitro. These results show that HSV-1 infection alters the in vitro transcriptional specificity of RNA polymerase II and demonstrate that this system should be useful for studying in vitro the regulation of gene transcription.