RNAs transcribed from a 3.6-kilobase SmaI fragment of the short unique region of the herpes simplex virus type 1 genome.

A 3.6-kilobase (kb) SmaI subclone of the BamHI J fragment of herpes simplex virus type 1 (KOS) DNA was utilized to characterize the mRNAs transcribed from the genome segment (0.91 to 0.93 map units) that encodes glycoprotein D mRNA. RNA blotting demonstrated two major RNA species of 2.3 and 1.5 kb. 5' and 3' mapping with 32P-end-labeled DNA fragments indicated that these RNAs are a nested set, each having its own promoter and 3' terminus. Less abundant RNA species with discrete 5' ends were also observed. Precise 5' mapping and sequence data located the initiation sites and demonstrated TATA boxes, CAT boxes, and AC-rich regions in the appropriate positions. 3' mapping located a common end for both mRNAs, but the 2.3-kb mRNA was reduced in size by splicing at a point near the RNA terminus. In vitro runoff transcription experiments confirmed the location of the two promoters and showed that an uninfected cell extract initiated faithfully at both sites. Despite the similarities in DNA structure and the apparent equal efficiency of promoter utilization in vitro, the 2.3-kb mRNA appeared in the cytoplasm early (1 h) after infection, whereas the 1.5-kb mRNA was delayed until 3 h after infection.