Herpes simplex virus type 2 mutants with deletions in the intergenic region between ICP4 and ICP22/47: identification of nonessential cis-acting elements in the context of the viral genome.

In herpes simplex virus type 2, the mRNAs of ICP4 and ICP22/47 are divergently transcribed and their transcription initiation sites are separated by 750 base pairs (L. J. Whitton and J. B. Clements, Nucleic Acids Res. 12:2061-2078, 1984). This 750-base-pair region contains many recognized cis-acting elements, including two TATA boxes, numerous Sp1-binding sites, four TAATGARAT motifs, at least one ICP4-binding site, and two origins of replication (oriS) linked in tandem. In this report, we describe the construction of mutant viruses with defined deletions that eliminate these elements either singly or in combination. The phenotypic properties of these mutants indicate that (i) the TAATGARAT motifs and their neighboring elements affect the levels of transcription of both ICP4 and ICP22/47 similarly, (ii) the TATA box serving ICP4 is required for efficient ICP4 mRNA synthesis and for determining the initiation site of transcription, (iii) the ICP4-binding site located at the start of ICP4 transcription is at least partially responsible for the decreased levels of ICP4 mRNA observed in the presence of immediate-early and early gene products, and (iv) mutants bearing deletions that eliminate the entire conventionally recognized ICP4 promoter generate sufficient ICP4 mRNA to maintain viability in cells not expressing ICP4. Additionally, our inability to generate viable deletion mutants lacking all copies of oriS suggests that at least one copy of oriS may be essential for virus replication.