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.

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 ICP4 (or Vmwl75) is synthesized during the immediate early phase of infection by herpes simplex virus and regulates the transcription of delayed early and late viral genes. We obtained a partially purified preparation of soluble ICP4 under nondenaturing conditions. Physical constants for native ICP4 were empirically determined by molecular sieve chromatography and sucrose density gradient ultracentrifugation. The Stokes radius of native ICP4 was 8.72 x 10-7 cm. The sedimentation coefficient of native ICP4 was 9.OOS. From these values, the calculated molecular weight of native ICP4 was 342,000, a value which is twice that of monomeric ICP4, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The failure of any other polypeptides to specifically coprecipitate with native ICP4 in the presence of anti-ICP4 antibody indicates that the 342,000-dalton complex is a homodimer of ICP4. The frictional coefficient ratio of native ICP4, which is 1.9, indicates that the homodimer is a highly elongated molecule.

The herpes simplex virus immediate-early gene product ICP4 activates the transcription of viral early and late genes. We characterized the DNA sequence elements of the early glycoprotein D (gD) gene that play a role in the response to ICP4 in vitro. Using gel mobility shift assays and DNase I footprinting, we identified three ICP4-binding sites, two 5' to the mRNA start site and a third within the coding region. Site II, which gave a footprint between nucleotides-75 and-111 relative to the RNA start site, was previously identified by Faber and Wilcox and contained the reported consensus ICP4-binding site. Site III, which was located between nucleotides + 122 and +163, was very similar to the site II sequence, including a core consensus binding sequence, TCGTC. The site I sequence (nucleotides-308 to-282), however, did not share significant homology with either site II or site III. In vitro transcription experiments from mutant constructs of the gD promoter indicated that all three ICP4-binding sites contribute to the stimulation of transcription by ICP4. DNase I footprinting of the gD promoter with uninfected nuclear extracts of HeLa cells showed protection of two very G-rich sequences between nucleotides-33 and-75. We propose that optimal transcription of the gD gene depends on the interaction of ICP4 with multiple binding sites across the gene and cellular factors that recognize specific sequence elements in the promoter.

ICP4 is a herpesvirus-encoded protein that is expressed during the immediate-early phase of productive infection and is required for efficient transcription of viral genes during the early and late phases of infection. Previous studies have shown that ICP4 is a component of specific protein-DNA complexes but have not revealed whether native ICP4 directly recognizes specific nucleotide sequences. Using DNA affinity chromatography, we have purified ICP4 to near homogeneity. The purified preparation consists primarily of dimeric and tetrameric forms of ICP4. As shown by DNase protection experiments, purified ICP4 binds directly to specific sequences in viral DNA. The stability of ICP4-DNA complexes is increased over 100-fold by shifting the temperature from 23 to 4°C. The equilibrium dissociation constant of ICP4-DNA complexes formed at 4°C in the presence of 100 mM NaCI was determined to be approximately 1.1 nM.