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A Specific 15-Bp TATA Box Promoter Element Is Required for Expression of a Herpes Simplex Virus Type 1 Late Gene

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A Specific 15-Bp TATA Box Promoter Element Is Required for Expression of a Herpes Simplex Virus Type 1 Late Gene Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press A specific 15-bp TATA box promoter element is required for expression of a herpes simplex virus type 1 late gene Fred L. Homa, 1,4 Joseph C. Glorioso, 2,3 and Myron Levine 1,s 1Department of Human Genetics, 2Unit for Laboratory Animal Medicine, 3Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618 USA The herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) gene is a true late or ~/2 gene in that its expression shows a strict requirement for viral DNA replication. Elements required for regulated expression of this gene were previously shown to consist of the gC TATA box, transcription start site and a large portion of the leader sequence of the gC gene. In this paper we show that transcription of the gC gene requires a 15-bp sequence, GGGTATAAATTCCGG, which contains the gC TATA box. This sequence contains specific promoter elements because replacement of this sequence with either the TATA box of the HSV-1 early thymidine kinase (tk) gene or two random TATA-Iike elements results in a transcriptionally inactive gC gene. In addition, we show that temporal expression of HSV 13 and ~/genes at early and late times during infection are controlled by separate and distinct regulatory elements; regulatory signals distal to the TATA box are needed for early expression, whereas a gC-like TATA box is needed for late expression. These signals were identified by construction of a chimeric HSV gene that contained the distal control signals of the ~ tk gene fused upstream of the TATA sequence of the ~/2 gC gene. When RNA was isolated at various times postinfection from cells infected with a virus whose genome contained this chimeric tk-gC gene, synthesis of gC mRNA showed both early and late kinetics. [Key Words: Herpes simplex virus type 1; glycoprotein C promoter; temporal gene expression; TATA box; recombinant viruses; deletion mutants] Received September 25, 1987; revised version accepted November 23, 1987. Herpes simplex virus type 1 (HSV-1) has a double- sion. Within this sequence are elements found in many stranded DNA genome of approximately 150 kb that en- eukaryotic promoters, including a proximal TATA box codes three general classes of genes: c~ or immediate element and distal elements such as Spl-binding sites early (IE), [3 or early, and ~/or late. These genes are tran- and CAAT box homologies (McKnight and Tjian 1986). scribed by the host cell RNA polymerase II, and expres- Located far upstream of all five IE genes are varying sion is largely regulated at the level of transcription (Go- numbers of enhancer like elements bearing the AT-rich dowski and Knipe 1986; Weinheimer and McKnight consensus sequence, TAATGARATTC (Mackem and 1987). The IE genes are transcribed first and are defined Roizman 1982a, b; Cordingley et al. 1983; Preston et al. as those HSV genes that are expressed in the absence of 1984). IE transcription is stimulated by a structural com- prior de novo protein synthesis (Honess and Roizman ponent of the virus particle Vmw65 (Post et al. 1981; 1974). IE transcripts can be detected at approximately Batterson and Roizman 1983; Cordingley et al. 1983; 0.5 hr postinfection and reach peak levels-at approxi- Campbell et al. 1984; Kristie and Roizman 1984; Preston mately 2-3 hr postinfection (Harris-Hamilton and Ba- et al. 1984; Dalrymple et al. 1985; Pellet et al. 1985). The chenheimer 1985; Weinheimer and McKnight 1987). TAATGARATTC elements are required for the stimula- The transcription of IE genes is controlled by physically tion by Vmw65, which suggests that IE gene expression separable and movable promoter and regulatory se- is controlled by either direct or indirect interaction of quences (Mackem and Roizman 1982). The promoter Vmw65 with this sequence (Kristie and Roizman 1987). component is found within 110 bp upstream of the tran- Transcription of early genes requires functional IE scription start site and is absolutely required for expres- gene products (Honess and Roizman 1975). Studies with temperature-sensitive and deletion mutants have shown that the product of the oL4 gene is essential for transacti- vation of early promoters (Preston 1979; DeLuca et al. 1984, 1985; DeLuca and Schaffer 1985). Early transcripts 4present address: Division of MolecularBiology, The Upjohn Company, Kalamazoo, Michigan 49001 USA. can be detected at approximately 2 hr postinfection, SCorrespondingauthor. reach peak levels at 5-6 hr postinfection, and gradually 40 GENES& DEVELOPMENT2:40-53 © 1988 by Cold Spring HarborLaboratory ISSN 0890-9369/88 $1.00 Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Transcriptional control of HSV late genes decrease to nearly undetectable levels late in infection cludes the gC TATA box, contains signals essential for (Harris-Hamilton and Bachenheimer 1985; Weinheimer fully regulated expression of the gC gene. We also show and McKnight 1987). The upstream regions of early that the TATA sequence of the tk gene and two random genes are less complex than those of IE genes in that TATA-like sequences are not capable of substituting for they lack enhancer like elements. The cis-acting signals the gC TATA sequence. In addition, evidence is pre- important for regulated expression of early genes have sented indicating that expression of [3 and ~/HSV-1 genes been shown to be located not more than 110 bp 5' of at early and late times during infection are controlled by early mRNA capsites (Zipser et al. 1981; Smiley et al. separate and distinct regulatory elements; the proper 1983; Halpern et al. 1984). Extensive mutational anal- DNA sequences distal to the TATA box are needed for ysis has identified four sequence elements required for early expression, whereas a gC-like TATA element is efficient expression of the HSV-1 early thymidine kinase needed for late expression. (tk) gene (McKnight et al. 1981; Zipser et al. 1981; McKnight and Kingsbury 1982; Smiley et al. 1983; Coen Results et al. 1986). These elements consist of a proximal TATA box and three upstream regions consisting of two Spl The gC promoter consists of the 15-bp sequence, binding sites separated by a CAAT box. Disruption of GGG TA TAAA TTC C G G any of these elements alters the level of tk expression. Transcription of late genes also requires functional IE We had previously described the construction of plasmid gene products and studies with temperature-sensitive pGC, which contained the entire 2.7-kb HSV-1 gC gene mutants have shown that the product of genes a4 and plus 1.3 kb of 5'-flanking sequence in a pUC18-based c,27 are essential for efficient expression of late pro- vector (Fig. 1). Using this plasmid, we constructed nine moters during infection (DeLuca and Schaffer 1985; De- deletion plasmids, pGCAI-pGCA9, in which varying Luca et al. 1985; Sacks et al. 1985). The late genes form portions of DNA were removed between bases -569 two subclasses, ~/1 and ~/2, differing in their dependence (BstEII site) and + 124 (BglII site) relative to the start of on viral DNA replication for expression. Prior to the gC transcription. These mutations were then transferred onset of viral DNA synthesis, 1-2 hr postinfection, low from plasmids into the viral genome through homolo- levels of ~/1 mRNAs can be detected while no ~2 tran- gous recombination to generate nondefective HSV-1 re- scripts are present (Holland et al. 1980). Following the combinants. The gC gene is amenable to these muta- onset of viral DNA replication, 2-3 hr postinfection, ex- tions because the gC-gene product is not required for pression of both ~1 and ~/2 transcripts increases and replication of HSV-1 in tissue culture (Heine et al. 1974; reaches peak levels at 7-8 hr postinfection and remains Holland et al. 1984a). Analysis of RNA extracted from at these high levels late in infection (Harris-Hamilton cells infected separately with each of the nine deletion and Bachenheimer 1985; Weinheimer and McKnight viruses showed that the DNA sequences required for 1987). regulated expression of this late HSV-1 gene lie within Recently we reported a study defining the cis-acting bases -34 to + 124 (Homa et al. 1986a). To further de- DNA sequences required for regulated expression of the fine the DNA sequences that comprise the gC promoter, glycoprotein C (gC) gene, a model ~/2 gene of HSV-1 six additional deletion viruses were constructed, (Homa et al. 1986a). Using a set of deletion mutant vi- A10-A15. These viruses each have deletions that re- ruses, each of which was missing varying portions of move varying portions of the -34 to + 124 sequence of DNA within bases - 569 to + 124 relative to the site of the gC promoter (Fig. 2) and were isolated by cotrans- initiation of the gC message, we showed that the DNA fecting the appropriate plasmid DNA (Fig. 1) with HSV sequences required for regulated expression of this ~/2 A2 viral DNA into Vero cells. The A2 virus contains a gene lie within bases -34 to + 124. Within the 34 bases deletion that removed bases -569 to + 124 of the gC upstream of the gC transcription start site is the TATA gene. The progeny from the transfection were then sequence located at -30. More recently, we have shown screened for the ability to hybridize with a restriction that the 63 bp between -34 and + 29 were sufficient for fragment specific for the region deleted in the a2 virus response to a-gene product activation in transient co- by an in situ hybridization screening procedure de- transfection assays (Shapira et al.
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