We report that the herpes simplex virus (HSV) transcription regulatory protein designated ICP4 is a component of a stable complex between protein and specific nucleotide sequences in double-stranded DNA formed by addition of exogenous DNA to either a crude extract obtained from HSV-1 infected cells or a partially purified preparation of native ICP4. DNA sites which are bound directly or indirectly to ICP4 have been designated ICP4/protein binding sites. Three independent ICP4/protein binding sites have been identified by DNAse footprinting; two are in the vector pBR322 and one is located approximately 100 nucleotides upstream from the HSV glycoprotein D mRNA cap site. Comparison of the nucleotide sequences in these three sites reveals several regions of homology. We propose that the sequence 5'-ATCGTCNNNNYCGRC-3' (N = any base; Y = pyrimidine; R = purine) forms an essential component of the ICP4/protein binding site.

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We report on phosphorylation, the stability of the bound phosphate, and the properties of several phosphorylated infected-cell polypeptides (ICPs) synthesized in cells infected with herpes simplex virus 1 and 2. Our results and conclusions are as follows. (i) Phosphorylation of ICPs occurs by at least two different pathways. Thus, the 4a and 4c electrophoretic forms of ICP 4 were labeled with 32P during a pulse concurrently with their synthesis, whereas ICP 22 and ICP 27 were labeled with 32p only during a subsequent chase in the presence of unlabeled phosphate. (ii) Pulse-chase studies with [35S]methionine and 32p indicate that whereas most polypeptides are stable, the bound phosphate with few exceptions

The HSV gene encoding ICP4 is negatively regulated and the HSV gene encoding thymidine kinase is positively regulated by ICP4 in vivo. We report that ICP4 is a component of a stable complex that contains protein and a sequence of approximately 28 nucleotides that span the ICP4 gene transcription initiation site. The association of ICP4 with DNA sequences between positions -103 and +32 relative to the ICP4 mRNA start site was demonstrated by DNA binding immunoassays. DNase footprinting revealed that nucleotides between positions -8 and +20 are protected by ICP4. In contrast, binding of ICP4 to sequences flanking the mRNA start site in the thymidine kinase gene was not observed. Models for ICP4-mediated positive or negative regulation of HSV gene transcription are discussed.