Herpes simplex virus immediate early infected-cell polypeptide 4 binds to DNA and promotes transcription.

Abstract

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 ...

Recruitment of Herpes Simplex Virus Type 1 Transcriptional Regulatory Protein ICP4 into Foci Juxtaposed to ND10 in Live, Infected Cells

“…The IE protein ICP4 is essential for viral infection because it is required to activate the transcription of the early and late genes (11,37). Consistent with its ability to bind to viral DNA (3,33), ICP4 is recruited into viral replication compartments containing large amounts of replicating viral DNA (10,28,38). However, prior to the onset of DNA replication, ICP4 in fixed cells has been observed to be distributed diffusely throughout the nucleus, with foci appearing as infection proceeds (10,28,38,46).…”

A Prominent Serine-rich Region in Vmw175, the Major Transcriptional Regulator Protein of Herpes Simplex Virus Type 1, is not Essential for Virus Growth in Tissue Culture

“…Vmw175 is a sequence-specific D N A -b i n d i n g protein which recognizes a number of different sequences of which m a n y contain the consensus A T C G T C (Faber & Wilcox, 1986b;Beard et al, 1986;Michael et al, 1988). The sequence at the cap site of the IE-3 promoter is strongly bound by Vmw175 (Mfiller, 1987).…”

The Initiator Element in a Herpes Simplex Virus Type 1 Late-Gene Promoter Enhances Activation by ICP4, Resulting in Abundant Late-Gene Expression

“…ICP4 is a nuclear phosphoprotein that acts as a homodimer to activate or repress transcription depending upon the promoter (12,15,26,54,63,67,71). Genetic and biochemical studies have shown that ICP4 binds to DNA and interacts with a component(s) of general RNA polymerase II transcription machinery to activate or repress transcription (2,8,17,29,42,64,65,84). ICP4 forms a tripartite complex with TFIIB and TATA binding protein (TBP) on DNA, interacts with TBP-associated factor 250 (8,84), and promotes the formation of transcription preinitiation complexes on promoters (28).…”