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.

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.

In herpes simplex virus (HSV)-infected cells, there is a sequential expression of viral genes. In vivo experiments have implicated the Mr 175,000 immediate early protein ICP4 (infected-cell polypeptide 4) in the regulation of viral RNA synthesis, but the mechanism whereby ICP4 regulates transcription of viral genes is at present unknown. In this report we describe experiments with an in vitro transcription system and a purified preparation of ICP4 (estimated 5% of total protein). Using DNA from the HSV glycoprotein D gene (gD) as the template, we have observed that (i) specific binding occurs between ICP4 and DNA sequences adjacent to the gD gene promoter and (il) ICP4 stimulates initiation of transcription from thegD gene. The degree of stimulation depends on the amount of ICP4 present in the incubation. The kinetics of RNA synthesis demonstrate that the protein acts at the initiation step of transcription. These results identify ICP4 as a viral transcription factor whose presence on DNA facilitates the formation of transcription complexes.Herpes simplex virus (HSV) proteins synthesized in infected cells change in both number and character during productive infection (1,2 We have used an in vitro transcription system (27) to investigate how a partially purified preparation of the viral protein ICP4 interacts with DNA from the early HSV gene for glycoprotein D (gD). In this paper we present evidence that ICP4 binds specifically to DNA sequences adjacent to the gD gene promoter and stimulates accurate transcription from this early gene. The mechanism of stimulation by ICP4 involves an increase in initiation of RNA synthesis. This report identifies a specific step in the transcription process that is regulated by an HSV protein.MATERIALS AND METHODS Template DNA. The HSV DNA used in this study was prepared from the plasmid pJB3. This plasmid contains the Sma I fragment subcloned from the BamHI fragment J of HSV type 1 (HSV-1) (KOS). The construction pJB3 and a simplified restriction map of the Sma I fragment are shown in Fig. 1. More details on the plasmid and its use in mapping the gD mRNA are presented in an earlier publication (36). To obtain the Ava I fragment 1 for use in the in vitro transcription reactions, plasmid DNA was purified by two cycles of cesium chloride centrifugation, cut with the restriction enzyme Ava I (Bethesda Research Laboratories), and extensively extracted with phenol/chloroform, 1:1 (vol/vol). The DNA fragments were precipitated with ethanol, redissolved in buffer, and separated by electrophoresis on 1% agarose gels. The 1.55-kilobase-pair (kbp) Ava I fragment 1 was isolated by electrophoresis into a block of low-temperature-gelling agarose, application of heat to 680C, extraction with phenol, and precipitation with alcohol. The DNA fragment was dissolved in 10 mM Tris chloride, pH 7.5/1 mM EDTA and was used directly as template for in vitro transcription.The Sst I (Sac I) subclone of pJB3 was constructed by inserting the Sst I fragment that contains the gD gene into the unique ...

The viral polypeptide ICP 4 (or Vmw 175) is synthesized during the immediate early phase of infection by herpes simplex virus (HSV) and is required during the viral reproductive cycle for efficient transcription of delayed early viral genes. Replication of mutant strains of HSV-1 such as tsLB 2 that encode a temperature-sensitive variant of ICP 4 does not proceed beyond the immediate early phase in cells that are infected and maintained at the nonpermissive temperature (NPT). Under these conditions, the immediate early viral polypeptides accumulate to levels that are 10 to 100 fold greater than normal. We have investigated the use of tsLB 2-infected cells maintained at the NPT as a source for substantial amounts of ICP 4 for further characterization. Extraction of ICP 4 from tsLB 2-infected cells requires 0.5 M NaCl and yields aggregates that contain ICP 4, ICP 6, ICP 27, and lesser amounts of other proteins. These large aggregates cannot be disrupted under nondenaturing conditions and thus are not a suitable source for native ICP 4. We have used this overproduced ICP 4 as an antigen to generate ICP 4-specific antibody and for characterization of the primary structure of ICP 4. Analysis of acid-hydrolysed 32P-labeled ICP 4 revealed that the major phosphorylated residues in ICP 4 are phosphoserine and phosphothreonine.