copies of a consensus response element commonly referred to as the 29,33,51,63), plus, in some cases, an overlapping consensus octamer element, ATGCTAAT (39; C. apRhys, D. M. Ciufo, E. A. O'Neill, T. J. Kelly, and G. S. Hayward, submitted for publication). In addition, 11 CCCGCCC elements, which appear to be associated with basal expression properties of the IE175-IE68 region, have been shown by DNA footprinting studies to bind to the purified cellular Sp-1 factor (25).During HSV infection, the IE175 gene product is essential for progression of the lytic cycle (8, 50, 60) and for activation of the transcription of the delayed-early (DE) class of viral genes (30,31,46,50). The isolated viral DE and late promoters, whether associated with their own genes (11,55) or as hybrid HSV promoter-driven reporter genes, also respond to virus superinfection in an IE175-dependent fashion, both when in an integrated state in permanent DNAtransfected cell lines (6,9,11,12,35,53) and in transientexpression assays with superinfecting virus (14,40). In DNA cotransfection assays, the isolated IE175 gene encodes a trans-acting factor that stimulates expression of HSV DE promoter targets (13,21,41,52) and inhibits expression from its own promoter in IE175- CAT (9,22,42,43). Although the HSV IE110 gene product was shown to stimulate expression 4307 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from with polynucleotide kinase and [-y-32P]ATP. The two com-J. VIROL.on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from DNA TARGET SITES FOR IE175 BINDING AND AUTOREGULATION plementary oligonucleotides were annealed by heating to 65°C in 50 mM NaCI-10 mM MgCl2-25 mM Tris hydrochloride (pH 8.0)-100 ,ug of bovine serum albumin per ml-2 mM P-mercaptoethanol followed by slow cooling. For cloning, the double-stranded oligonucleotides were ligated after phosphorylation with polynucleotide kinase to BamHI-plus-BglII-cleaved pGH59 DNA. The resulting plasmids, pGH123a and pGH123b, contained sequences from -8 to