Virology 272, 191–203 (2000) doi:10.1006/viro.2000.0365, available online at http://www.idealibrary.com on

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Two Leaky-Late HSV-1 Promoters Differ Significantly in Structural Architecture

Pauline T. Lieu and Edward K. Wagner1

Program in Animal Virology, Department of and Biochemistry, University of California, Irvine, Irvine, California 92697-3900 Received February 16, 2000; returned to author for revision March 23, 2000; accepted April 20, 2000

The HSV-1 VP5 and VP16 transcripts are expressed with leaky-late (␥1) kinetics and reach maximal levels after viral DNA replication. While the minimal VP5 includes only an Sp1 site at Ϫ48, a TATA box at Ϫ30, and an initiator (Inr) element at the cap site, here we show that elements upstream of Ϫ48 can functionally compensate for the mutational loss of the critical Sp1 site at Ϫ48. To determine whether this is a general feature of leaky-late promoters, we have carried out a detailed analysis of the VP16 promoter in the context of the viral genome at the gC locus. Sequence analysis suggests a great deal of similarity between the two. Despite this, however, mutational analysis revealed that the 5Ј boundary of the VP16 promoter extends to ca. Ϫ90. This region includes an Sp1 binding site at Ϫ46, CAAT box homology at Ϫ77, and “E box” (CACGTG) at Ϫ85. Mutational and deletional analyses demonstrate that the proximal Sp1 site plays little or no role in promoter strength; despite this it can be shown to bind Sp1 protein using DNA mobility shift assays. Like the VP5 promoter, the VP16 promoter also requires an at the cap site. The VP16 Inr element differs in sequence from that of the VP5 promoter, and its deletion or mutation has a significantly smaller effect on promoter strength. The difference between these two Inr elements was confirmed by our finding that the VP16 initiator element binds to the 65-kDa YY1 factor, and the VP5 Inr element competes poorly for the binding between the VP16 element and infected cell proteins in comparative bandshift assays. While the VP16 Inr sequence is identical to that of several murine TATA-less promoters, the VP16 Inr requires a TATA box for measurable activity. © 2000 Academic Press

INTRODUCTION (Huang and Wagner, 1994; Homa et al., 1991; Guzowski and Wagner, 1993). Regulated gene expression during the productive rep- Much of the control of expression of these various lication cycle of herpes simplex virus type 1 (HSV-1) has classes of viral proteins is at the level of transcription, been extensively reviewed (Wagner, 1999; Wagner et al., and we and others have demonstrated that both the 1998; Jameson et al., 1998; Hay and Ruyechan, 1992). kinetics and the levels of expression of a particular Viral proteins furnishing the functions necessary for viral transcript encoding each viral protein are dictated by gene expression, replication, and viral assembly are ex- pressed in a coordinated and sequential manner. Two features of the promoter controlling that transcript groups, the immediate-early or ␣ proteins required for (Huang and Wagner, 1994; DeLuca and Schaffer, 1985; activating the expression viral genes and the early or ␤ McGeoch, 1991; Dennis and Smiley, 1984). The functional proteins involved genome replication reach their maxi- architectures of early and late promoters have been mum abundance prior to high levels of viral DNA repli- carefully characterized, and, while they share some of cation (Daksis and Preston, 1992; Everett, 1984; Jameson the general features with the majority of eukaryotic pol II et al., 1998; Wagner et al., 1995). As this occurs, the promoters, they clearly differ from each other (Corden et relative abundance of these early proteins declines and al., 1980; Fantino et al., 1992; McKnight and Kingsbury, late proteins predominate. Many late proteins are in- 1982; McKnight and Tjian, 1986). Indeed, a TATA box Ϫ Ϫ volved in virus morphogenesis and maturation. These, located between 25 and 30 relative to the cap site is too, can be divided into two subclasses: leaky-late (␥1) the most constant defining feature of HSV promoters. Its and strict-late (␥2). While the maximal expression of late sequence and position relative to the cap site and other genes is dependent upon viral DNA replication, the ␥1 cis-acting elements are intimately involved in controlling group is expressed at appreciable levels prior to the the levels, but not kinetics, of transcription (Homa et al., initiation or in the absence of viral DNA synthesis while 1988; Homa et al., 1986; Flanagan et al., 1991; Pande et the ␥2 group is readily detectable only upon such syn- al., 1998). thesis. Thus, expressions of this latter class of proteins Early and late promoters also contain other cis-acting are particularly sensitive to inhibitors of DNA replication elements involved in interaction with cellular transcrip- tional machinery, but the arrangement and exact nature of these elements differ markedly in a class-specific way. 1 To whom correspondence and reprint requests should be ad- Early promoter architecture is characterized by the pres- dressed. Fax: (949) 824-8551. E-mail: [email protected]. ence of one or more sequence motifs binding cellular

0042-6822/00 $35.00 191 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 192 LIEU AND WAGNER transcription factors (such as Sp1, CAAT, and Hinf-P) however, they differ in both sequence and contribution to upstream of the TATA box. With the exception of the promoter strength. The difference between these two Inr repressive strong ICP4 binding site in some immediate- elements was confirmed by our finding that the VP16 early promoters, there are no cis-acting elements down- initiator element binds to the 65-kDa YY1 transcription stream of the TATA box. In contrast, late promoters share factor. In contrast, we have previously demonstrated that a requirement for an initiator (Inr) element at the cap site the VP5 initiator element binds to a 35-kDa cellular pro- (Mavromara-Nazos and Roizman, 1989; Pande et al., tein (Huang et al., 1996). 1998; Huang and Wagner, 1994; Huang et al., 1993). Thus, while our results confirm our model that ␥1 The two subclasses of late promoters also differ mark- promoters have architectural features distinct from both edly. A number of ␥2 promoters require no elements early and strict-late promoters, they also reveal consid- upstream of the TATA box, but contain an element lo- erable variation within this class. While the biological cated 20 to 30 bases downstream of the cap site. Re- significance of the differences in late promoter architec- cently, we showed that this downstream activation se- ture is not entirely clear, knowledge of the range of quence or DAS is involved in stabilization of the preini- possible variations within a given kinetic class-specific tiation complex mediated by DNA–protein interactions architectural arrangement is important in understanding (Petroski and Wagner, 1998). In contrast, our studies on this and in designing useful vectors and viral reagents the VP5 promoter and the studies of others on the gE and for introducing genes into specific tissues. gB promoters indicate that ␥1 promoters require tran- scription factor binding sites upstream of the TATA box in RESULTS addition to an Inr sequence (Guzowski et al., 1994; Huang and Wagner, 1994; Sethna and Weir, 1993). The VP5 promoter contains both core and redundant In this paper, we extended our studies by performing a elements thorough structural comparison between the VP5 pro- We previously determined that despite the richness in moter and that controlling expression of another ␥1 the cis-acting elements of the VP5 promoter (shown in gene, VP16. We have previously shown that critical ele- Fig. 1), the only critical elements required for full pro- ments of the VP5 promoter extend only from the Inr moter expression are the Sp1 site at Ϫ48, the TATA box element through an Sp1 site at Ϫ48 (Huang et al., 1993; at Ϫ30, and the Inr element at the cap site (Huang et al., Huang and Wagner, 1994). Despite this, sequence anal- 1993; Huang and Wagner, 1994). Mutations of the Sp1 ysis shows that this promoter is rich in cis-acting ele- site at Ϫ48 in the context of the truncated promoter result ments further upstream including Sp1 binding sites at in a major reduction in (Fig. 1, lane iii). Ϫ144, Ϫ208, and Ϫ268; a CAAT binding site at Ϫ111; However, when the mutated Sp1 site at Ϫ48 was put in and a YY1 factor binding site at Ϫ78. In this report, we the context of the upstream YY1 site at Ϫ78 or the CAAT show that elements upstream of Ϫ48 can functionally box at Ϫ111, promoter activity of both recombinant vi- compensate for the mutational loss of the critical Sp1 ruses was essentially the same as that of the full-length site at Ϫ48. promoter (Fig. 1, lanes v and vi). Representative experi- Sequence analysis of the VP16 promoter suggests a ments are shown in Fig. 1, as are quantified results of great deal of similarity with the VP5 promoter in terms of several replicated determinations. This result demon- the arrangement of binding sites (in- strates that cis-acting elements upstream can compen- cluding Sp1 sites at Ϫ133, Ϫ123, and Ϫ46). Despite such sate for the loss of the downstream Sp1 site at Ϫ48 and similarities, mutational and deletional analyses in the that this promoter displays a high degree of redundancy context of the viral genome at the gC locus revealed that in sequence elements. none of the Sp1 sites are required for promoter activity, and the 5Ј boundary of the VP16 promoter extends to ca. Functional architecture of the VP16 promoter Ϫ100. The essential region includes a CAAT box homol- ogy at Ϫ77 and an “E box” (CACGTG; Kiermaier et al., The VP16 transcript is expressed with leaky-late kinet- 1999; Roy et al., 1997; Luo and Sawadogo, 1996) at Ϫ85. ics—equivalent to the kinetics of expression of the VP5 This latter site has the potential to bind a number of transcript. A survey of recognizable cis-acting elements tissue-specific regulatory factors including USF (Kier- in the VP16 promoter is shown in Fig. 2A and demon- maier et al., 1999; North et al., 1999; Roy et al., 1997; Luo strates a cap site located 30 bp downstream of a TATA and Sawadogo, 1996). Some other cis-acting feature of homology region (Ϫ30 TTAAAT-25) (Blair et al., 1987), a the promoter lying between this E-box and the Sp1 site at number of potential Sp1 binding sites, a CAAT box, and Ϫ46 is also important for full activity, but there is no an E box (CACGTG). In order to assess functional simi- obvious transcription factor binding site homology within larities between the promoters controlling these two it. As noted, the Sp1 site at Ϫ46 contributes minimally to transcripts, we carried out an extensive study of the promoter activity, and, like the VP5 promoter, the VP16 functional architecture of the VP16 promoter. Specific promoter requires an initiator element at the cap site; promoter alterations are shown in Fig. 2A, as are quan- LEAKY-LATE HSV-1 PROMOTER ELEMENTS 193

FIG. 1. The effect of mutation of the critical Sp1 site at Ϫ48 on VP5 promoter activity in the context of upstream elements. (Top) The schematic representation of potential cis-acting VP5 promoter elements upstream of the cap site is shown. Despite these elements, the minimal promoter extends only to Ϫ48. Relative activity of the VP5 promoter within the context of 364 bases upstream and 63 bases downstream of the cap site is shown and is compared to that of a truncated promoter containing only 48 bases upstream of the cap site. The activity of promoters in which the Sp1 site at Ϫ48 was mutated in the context of various amounts of upstream sequence is also shown. Values are based on a minimum of three determinations per virus isolate using PhosphoImager analysis as described under Materials and Methods. Values are Ϯ20% of those shown. In the truncated mutant, the Sp1 site at Ϫ48 was changed from Ϫ48-GGGGTGGGGCGG-37 to Ϫ48 GGGGTGGttCGG-37. In the Ϫ86 and Ϫ137 mutants, the sequence was changed to Ϫ48 GGGGaGGttCGG-37. (Bottom) Representative primer extension experiments are shown. Rabbit skin fibroblasts were infected with a m.o.i. of 5 PFU/cell, total RNA was isolated at 7 h postinfection, and 10 ␮g of RNA was analyzed by primer extension. VP5 reporter gene expression detected with a primer binding within the ␤-galactosidase gene was compared to expression of wt VP16 transcripts used as internal control. tified measures of relative activity in expressing reporter of leader sequences to ϩ6 had no effect on expression ␤-galactosidase mRNA in recombinant viruses. These (lanes i to iii); this demonstrated that there was no ele- numbers are based upon a minimum of three replicate ment downstream of ϩ6 important for the activity of this experiments. promoter. We similarly defined the 5Ј extent of the VP16 pro- Deletion analysis moter. Since deletion analysis demonstrated that this ϩ Ј promoter extends no further than 6, all upstream dele- We first generated nested deletions to establish the 3 ϩ and 5Ј limits of the contiguous promoter. The 3Ј extent of tions were analyzed in a reporter extending to 6, rela- the promoter was determined in the context of a pro- tive to the cap site. The various promoter deletions con- ␤ moter extending to Ϫ276. Deletions to ϩ95, ϩ32, and ϩ6 trolling the reporter -galactosidase gene were recom- were generated and then inserted 5Ј of the reporter bined into the HSV genome and expression was ␤-galactosidase gene and recombined into virus as de- measured by analysis of reporter RNA levels again at 7 h scribed under Materials and Methods. Duplicate, inde- postinfection. Deletion to Ϫ100 had no significant effect pendent isolates were tested for each construct. Each on reporter expression (Fig. 2B, lane vii), but a deletion to recombinant virus was used to infect rabbit skin cells, Ϫ82 resulted in a reproducible decrease in reporter and infected cell mRNA was isolated at 7 h postinfection. mRNA levels to about 60% of control levels (lane v). This Typical results are shown in Fig. 2B. The expression of region contains a canonical E box (CACGTG) (Kiermaier reporter ␤-galactosidase mRNA was measured by et al., 1999; Roy et al., 1997; Luo and Sawadogo, 1996) primer extension with a reporter-specific primer. Deletion between Ϫ90 and Ϫ85. 194 LIEU AND WAGNER LEAKY-LATE HSV-1 PROMOTER ELEMENTS 195

Deletion of sequences between Ϫ82 and Ϫ52 re- tween the wt and Ϫ82 promoters is a result of the loss of sulted in a further threefold drop in reporter gene expres- the E box. sion (lanes xii and xiii). Survey of the sequence between One possible reason for the lack of observable effect Ϫ82 and Ϫ52 (5Ј-GGAGCCAATGTGGGGGGAAGTCAC- of the Sp1 sites identified by sequence was that they are GAGGTA-3Ј) revealed no readily recognizable transcrip- not functional. Therefore we investigated whether these tion factor binding site downstream of the CAT box. sites did, indeed, bind authentic Sp1. We used DNA Surprisingly, deletion of the Sp1 site at Ϫ46 causing a bandshift assays with purified epitope-tagged Sp1 and truncation to Ϫ34 resulted in no significantly greater loss with commercial HeLa nuclear extracts and Sp1 antibody of activity (not shown). Such results suggest that the for this study. Oligonucleotides corresponding to se- VP16 promoter contains a minimum of two separate quences from Ϫ61 to Ϫ33 or Ϫ138 to Ϫ111 containing wt cis-acting elements upstream of the TATA box. It is no- or mutant Sp1 sites were incubated with commercial table that, unlike the situation with the VP5 promoter, the Sp1. Data shown in Fig. 3A demonstrate that both oligo- putative Sp1 site at Ϫ46 is not sufficient for full promoter nucleotides containing the wt sites were efficiently activity. bound (lanes i and iv), but mutation of the Sp1 binding motifs in these oligonucleotides sites reduced binding to Mutational analysis of cis-acting elements upstream undetectable levels (lanes ii and iii). We used a commer- of the VP16 promoter cial HeLa cell extract and antiserum to Sp1 to further examine the relative binding of the two Sp1 motifs be- We next used mutational analysis to investigate tween Ϫ133 and Ϫ123 (Fig. 3B). Again, oligonucleotides whether the putative cis-acting elements upstream of the containing wt elements bound Sp1 efficiently (lanes ii minimal core had any role in the function of the subject and iv), and mutation of the site at Ϫ46 or Ϫ133 reduced promoter. Representative data are again shown in Fig. this binding markedly (lanes i and iii). In contrast, muta- 2B and summarized in Fig. 2A. We altered the promoter tion of the site at Ϫ123 had a less marked effect upon Ϫ Ϫ Ј sequence between 51 and 38 from 5 -GGGGCGGC- binding (lane v). The presence of the antibody markedly Ј Ј Ј CGTGCG-3 to 5 -GctgcaGCCagaCG-3 and inserted the supershifted the complex binding to the Sp1 sites at mutant sequence into the context of the VP16 promoter Ϫ133/Ϫ123 (lane viii). Again, no binding was seen when Ϫ ϩ extending from 276 to 6 to construct a reporter virus, the site at Ϫ133 was mutated (lane vii), but some binding and reporter RNA was measured by primer extension remained upon mutation of the site at Ϫ123 (lane ix). A analysis. The recombinant virus exhibited the same level similar supershift was observed with the antibody and of reporter gene expression as that of the wild-type the oligonucleotide containing the Sp1 site at Ϫ46 (not sequence (lane ix). Thus, the function of this Sp1 site has shown). no measurable role in promoter activity whether sites further upstream are present or not. Again, and as ex- Mutational analysis of the VP16 cap sequences pected from our deletion studies, mutations of two po- tential Sp1 binding sites at Ϫ123 and Ϫ133 from 5Ј- As discussed in the Introduction, where studied, HSV ACTGGGTGGCGGTGGGGTGGGGTT-3Ј to 5Ј-ACTG- promoters active at high levels following genome repli- GcagctgGTGatccGGGGTT-3Ј in the context of wt cation feature a cis-acting Inr-like element at or near the sequences downstream had only a minimal effect on transcript start site. We used mutational and deletion promoter activity (lane x). analyses to demonstrate that the initiator of the VP16 We also mutated the CAAT box sequence from Ϫ79 to promoter also has a significant role in its activity (results Ϫ70 (5Ј-AGCCAATGTG-3Ј to 5Ј-AGaagcaGTG-3Ј)inthe quantitated in Fig. 2A). Essentially, the 3Ј VP16 promoter context of a VP16 promoter extending to Ϫ82. Reporter cap sequence was deleted to Ϫ15. This was then used gene expression was essentially equivalent to that seen for insertion of a linker and wt or mutated cap sequences with the unmodified deletion to Ϫ82 (compare lanes v were then built back into the promoter. Modified promot- and vi). This indicates that the difference in activity be- ers were then tested for activity in recombinant reporter

FIG. 2. The functional extent of the VP16 promoter. (A) Promoter elements predicted from sequence analysis and modifications described in the text are shown here. Relative activity of the VP16 promoter within the context of 276 bases upstream and 95 bases downstream of the cap site is shown and compared to that of modified promoters shown. Values are based on a minimum of three determinations per virus isolate using PhosphoImager analysis as described under Materials and Methods. Values are Ϯ20% of those shown. Mutation of the Sp1 site at Ϫ46 came from changing Ϫ51-GGGGCGGCCGTGCG-38 to Ϫ51-GCTGCAGCCAGACG-38. Mutation of the Sp1 sites at Ϫ133 and Ϫ123 was done by changing Ϫ135-ACTGGGTGGCGGTGGGGTGGGGTT-113 to Ϫ135-ACTGCAGCTGGGGATCCGGGGTT-113. The cap site deletion mutant contains an AvaI linker inserted between Ϫ15 and Ϫ9. This results in changing Ϫ15-CGACCACGG-7 to Ϫ15-CTCCCCCGG-7. Alteration of the Inr sequence changed Ϫ4-TGTCATTCϩ4toϪ4-TAGTGCGTCϩ4, alteration of the TATA box changed Ϫ30-TTAAATGC-23 to Ϫ30-TCGAGTGC-23. (B) Representative primer extension analyses. (Top) Reporter gene expression; (bottom) products with primers for wt VP16 or gD transcripts used as internal control. The arrows in lanes i, ii, iii, and xv indicate the correct size for the extension product. 196 LIEU AND WAGNER

FIG. 3. Demonstration of Sp1 binding to identified sites in the VP16 promoter. (A) Commercial purified Sp1 was incubated with 32P-labeled double-stranded oligonucleotides containing Sp1 sites in the VP16 promoter. Lanes i and ii contain oligonucleotides corresponding to the mutant and wt Sp1 sites at Ϫ46 (see the legend to Fig. 2) in the context of Ϫ61 to Ϫ33. Lanes iii and iv are oligonucleotides with sequences from Ϫ138 to Ϫ111 containing the Sp1 sites at Ϫ123 and Ϫ133 (Ϫ133/Ϫ123 or a mutation at Ϫ133 (m-133/Ϫ123). The actual sequences are shown in the legend to Fig. 2. Material that did not enter the gel is seen in the wells at the top of the gel. (B) Sp1 containing HeLa nuclear extracts and Sp1 antibody in the Nushift Kit (purchased from Geneka Biotechnology, Inc.) were incubated with the same probes and fractionated on the same gel. Lanes i and ii contain oligonucleotides corresponding to the mutant and wt Sp1 sites at Ϫ46, and lanes iii and iv are oligonucleotides with sequences from Ϫ138 to Ϫ111 containing the Sp1 sites at Ϫ123 and Ϫ133 (Ϫ133/Ϫ123 or a mutation at Ϫ133 (m-133/Ϫ123). Lane vi (Std) contains oligonucleotides containing an Sp1 site supplied by the manufacturer. Lanes vii, viii, and ix contain the wt and mutant Sp1 sites in the Ϫ138 to Ϫ111 oligonucleotides as well as the anti-Sp1 antiserum supplied in the kit. virus. Primer extension analysis of reporter RNA isolated the presence of a mutated Inr sequence in the context from cells at 7 h postinfection demonstrated that a full of Ϫ276 resulted in undetectable reporter gene ex- deletion or an alteration of the cap sequences between pression (Figs. 2A and 2B, lanes xx and xxi). Ϫ4 and ϩ4 from 5Ј-TGTCATTC-3Ј to 5Ј-TGgtAcTC-3Ј re- duced activity by approximately 40% (lanes xv to xviii). The VP16 Inr specifically interacts with a cellular The DNA sequence around the VP16 transcript start protein site is identical to the Inr elements of the murine terminal deoxynucleotidyltransferase (TdT) and ribo- We next investigated specific interactions between nucleotide reductase promoters (Ϫ2-TCATTCϩ3), and uninfected and infected cell proteins and the VP16 Inr is quite similar to that of the adenovirus major late element by competitive gel shift analysis (Fig. 4). When 32 promoter (AdML; Ϫ2-TCACTϩ3). Both murine promot- P-labeled dsDNA probe containing the VP16 sequence ers lack a consensus TATA element, and the initiator from Ϫ15 to ϩ6(5Ј-CTCCCCCGGGCTGTCATTCCTC- has been shown to be able to mediate transcription by CGGGG-3Ј) was incubated with uninfected nuclear ex- direct interaction with the TFIID preinitiation complex tracts, a single major DNA–protein complex was de- (Smale and Baltimore, 1989; Kong et al., 1999; Johan- tected. Addition of increasing amounts of unlabeled sson et al., 1995). Accordingly, we wanted to determine probe DNA demonstrated full competition (lanes marked whether the VP16 initiator could function in the ab- “Self mock”), suggesting a specific interaction. Incuba- sence of the TATA box. We altered the VP16 promoter tion of labeled probe with infected cell extracts gave a sequence between Ϫ30 and Ϫ23 from 5Ј-TTA- more complex pattern with three complexes readily de- AATGC-3Ј to 5Ј-TcgAgTGC-3Ј and generated reporter tectable. In these extracts, however, only the complex recombinant viruses. Mutation of the TATA alone or in migrating with the same rate as that seen in uninfected LEAKY-LATE HSV-1 PROMOTER ELEMENTS 197

FIG. 4. Electrophoretic mobility shift assay of the binding of VP16 Inr element to cellular proteins. Nuclear extracts from mock-infected or HSV-1-infected rabbit skin fibroblasts were obtained as described under Materials and Methods. Extracts were incubated with the 32P-labeled double-stranded oligonucleotides of the VP16 promoter extending across Ϫ15 (5Ј-CTCCCCCGGGCTGTCATTCCTCCGGGG-3Ј)ϩ6. Competition was performed with unlabeled wt VP16 probe, the mutated VP16 initiator element Ϫ15 (5Ј-CTCCCCCGGGCTAGTGCGTCTCCGGGG-3Ј)ϩ6, the VP5 probe extending across Ϫ11 (5Ј-GATCCCCCGGGTCTGTTGGGGACACT-3Ј) ϩ15, or a double-stranded oligonucleotide containing the canonical Sp1 site and flanking bases (5Ј-ATTCGATCGGGGCGGGGCGAGC-3Ј). extracts was efficiently competed with an excess of self- promoters can be readily defined by deletion analysis, unlabeled probe (“Self Inf”). mutation of a critical VP5 promoter core element in the Further evidence of specific interaction was seen by context of the upstream elements results in little observ- inefficient competition for binding with unlabeled ds oli- gonucleotides with sequence corresponding to the mu- tated Inr element shown above to reduce promoter ac- tivity (“mInr mock” and “mInr Inf”). Significantly, the inef- ficient competition was especially notable in the infected cell extracts. In addition, unlabeled ds oligonucleotides with sequences corresponding to the wt VP5 Inr extend- ing from Ϫ11 to ϩ15 (5Ј-GATCCCCCGGGTCTGTTGGG- GACACT-3Ј) competed as poorly as the wt VP16 Inr sequence (“VP5Inr Inf”). The unlabeled ds oligonucleo- tide containing the Sp1 binding site (5Ј-ATTC- GATCGGGGCGGGGCGAGC-3Ј) did not compete at all for binding when used as an unlabeled competitor (Sp1 mock).

The VP16 initiator element interacts with YY1 protein The VP16 initiator element contains a putative binding site for YY1 (“CATT”), and we wanted to determine whether the YY1 protein binds to this site. We utilized 32P-end-labeled probes containing the VP16 Inr from Ϫ15 to ϩ6 with purified GST–YY1 fusion protein in another electromobility shift assay shown in Fig. 5. The ability of this protein to bind YY1 containing DNA has been re- ported elsewhere (Bennett et al., 1999). The results indi- cate that the recombinant YY1 protein binds to the wt FIG. 5. Electrophoretic mobility shift assay of the VP16 promoter Inr VP16 Inr fragment (lane i) and failed to bind to the mutant incubated with purified GST–YY1 fusion protein. Samples of 50 ng of GST–YY1 fusion protein (obtained from Dr. Tim Osborne (Bennett et al., VP16 Inr or VP5 Inr (lanes ii and iii). 1999)) were incubated with the 32P-labeled double-stranded oligonu- cleotides of the VP16 promoter extending over Ϫ15 (5Ј-CTC- DISCUSSION CCCCGGGCTGTCATTCCTCCGGGG3Ј)ϩ6, the mutated VP16 initiator element Ϫ15 (5Ј-CTCCCCCGGGCTAGTGCGTCTCCGGGG-3Ј)ϩ6, or The first important observation reported here is that the VP5 promoter extending across Ϫ11 (5Ј-GATCCCCCGGGTCTGTT- while the core cis-acting elements of the VP5 and VP16 GGGGACACT-3Ј)ϩ15. 198 LIEU AND WAGNER able effect. This is an important, if generally unrecog- protein YY1, while the VP5 Inr does not. Again the func- nized, consideration in assessing the functional extent of tional contributions of the elements are significantly dif- promoters in the context of the viral genome. For exam- ferent. Loss of the VP16 Inr results in about 40% of ple, linker-scanning mutagenesis of the gE promoter has maximal activity (Fig. 2), and we observed that this con- been interpreted as demonstrating that no cis-acting tribution to overall activity was the same at3hasatlate elements upstream of the TATA box are required (Sethna times (data not shown). Thus, the VP16 Inr appears to and Weir, 1993), but such results ignore the possible have little influence on the overall kinetics of expression presence of other cis-acting elements compensating for of this transcript. In contrast, alteration of the Inrs of the the mutated site(s). Clearly, both mutation and deletion VP5 and several other late HSV promoters has at least a must be used to fully characterize any specific promoter. fivefold effect on late promoter activity (Huang et al., The VP16 and VP5 promoters share similarities in 1993, 1996; Woerner and Weir, 1998; Huang and Wagner, sequence along with striking differences in functional 1994; Steffy and Weir, 1991). Also, in the case of the VP5 architecture. A major difference between the two promot- promoter, the effect of loss of the Inr on activity at early ers studied can be found in the extent and nature of the times is significantly less, which has the result of oper- core promoter elements identified. The deletion survey of ationally converting this promoter to a weak early pro- the 5Ј extent of the VP16 promoter summarized in Fig. 2 moter. This suggests that the roles of the Inr elements in demonstrates the requirement for sequences up to the two promoters differ mechanistically. Ϫ100. We were surprised that the VP16 promoter con- A major feature of the VP16 Inr is the presence of the tained functional Sp1 sites that had no role in measured YY1 binding site. While it has been shown that YY1 promoter activity and that there is at least one, as yet protein binds to several late HSV-1 promoters, including unidentified, element between the E box (CACGTG) and the VP5 and gD upstream of the TATA box (Mills et al., the Sp1 site at Ϫ46 that contributes significantly to pro- 1994), this is the first report of its occurrence near an moter activity. It is clear that the lack of function of the HSV initiator element; thus, it was important to confirm Sp1 sites in the Vp16 promoter is not due to any func- that YY1 actually binds the VP16 Inr. This interest was tional lack of transcription factor. This follows from the intensified by the varied effects this protein is known to fact that we have previously shown that functional Sp1 is have on transcription. YY1 is a multifunction protein that present in extracts of cells late after infection by HSV-1 can take part in repression or activation of transcription, (Huang and Wagner, 1994). It was shown there and here depending on the promoter context and other factors that the Vp5 promoter requires an Sp1 site for full activity present. It has been shown to interact with a number of late after infection; this underscores the presence and regulatory proteins: TBP, TFIIB, Sp1, TAFII55, and E1A availability of functional transcription factor. The E box (Bushmeyer et al., 1995; Thomas and Seto, 1999; Usheva between Ϫ85 and Ϫ90 in the VP16 promoter is the first and Shenk, 1996; Seto et al., 1991). Margolis et al. (1994) documentation of its functional occurrence in an HSV-1 reported that YY1 recognizes the initiator region of the promoter. This motif is found in numerous promoters HIV-1 LTR and decreased LTR-directed gene expression. including those occurring in HIV LTR and Epstein–Barr Expression of YY1 protein with an HIV-1 clone resulted in virus ED-L2 early lytic cycle promoter (Jenkins et al., decreased virus production in vivo. In contrast, the ade- 1997; Suzuki et al., 1999; Moriuchi et al., 1999). Proteins novirus E1A protein can alter YY1 activity so that instead binding to E boxes such as upstream stimulating factors, of functioning as a in the AdML promoter, its Kruppel-like factors, basic helix-loop-helix proteins, ke- binding activates transcription (Bushmeyer et al., 1995; ratinocyte-specific factors, and the HTLV-1 Tax protein Seto et al., 1991). Further, YY1 typically represses the have been shown to have tissue-specific regulatory adeno-associated virus P5 promoter, but in the presence roles. A number of reports have also shown that this of E1A protein, it can be converted into a potent tran- element has a role in tissue-specific promoter expres- scriptional activator. Seto et al. (1993) have shown that sion. Such tissue includes skeletal muscles, squamous the YY1 protein can bind to the adeno-associated virus epithelial cells, and nerve cells (Esser et al., 1999; type 2 P5 promoter initiator region and mediate transcrip- Wheeler et al., 1999; North et al., 1999; Moriuchi et al., tion through an initiator element in vitro. Subsequently, 1999; Suzuki et al., 1999; Jenkins et al., 1997; Athanikar Usheva and Shenk (1996) demonstrated that YY1 inter- and Osborne, 1998). It will be interesting to determine acts with TFIIB and RNA pol II and supports basal tran- whether the element has any role in HSV’s ability to scription from supercoiled plasmid templates in vitro, in replicate in various tissues. the presence of TFIIB and RNA pol II alone. It will be of The presence of an Inr element in the VP16 promoter interest in the future to study the role that the YY1 protein is expected from its general occurrence in late promot- plays in regulating HSV-1 viral gene expression. It will be ers, but the VP16 Inr sequence (Ϫ5-GCTGTCATTCCϩ5) of interest to determine whether ICP4, an essential pro- differs markedly from that of the VP5 Inr (Ϫ5- tein required for activating HSV gene expression, can CCCGGGTCTGTϩ5). Such differences are also con- interact with YY1 and influence its function. firmed by the fact that the VP16 Inr binds to the cellular The VP16 Inr sequence (Ϫ5gctgTCATTCcϩ5) shares a LEAKY-LATE HSV-1 PROMOTER ELEMENTS 199 hexanucleotide sequence with the murine TdT and ribo- and VP16 promoter in the gC locus using our standard nucleotide reductase R1 Inr elements (Ϫ5acgTCATTC- methods described in detail elsewhere and outlined be- gaaϩ5). Both mouse promoters lack a consensus TATA low (Lieu et al., 2000; Wagner et al., 1998; Guzowski et al., box, but we found that mutations of the VP16 TATA box in 1994; Huang and Wagner, 1994; Huang et al., 1993). the presence of the wt Inr sequences resulted in unde- tectable reporter gene expression. Thus, unlike the TATA- Mutations of the VP5 promoter elements less promoters, the VP16 Inr requires a functional TATA Mutations of the Sp1 site at Ϫ48 were generated in the box to mediate transcription. The requirement for a TATA context of the VP5 promoter containing sequences from box has been shown to be the mechanism of ICP4- Ϫ48 to ϩ63, Ϫ79 (containing the YY1 site) to ϩ63, and mediated transcriptional activation (Gu and DeLuca, Ϫ133 (containing both YY1 and the CAAT box) to ϩ63, 1994; Carrozza and DeLuca, 1998; Imbalzano and De- driving the expression of the bacterial ␤-galactosidase Luca, 1992; Kuddus et al., 1995; DeLuca and Schaffer, gene. Plasmids pCH204, pCH203, and pCH207 de- 1988). Since the HSV transcriptional activator ICP4 inter- scribed in Huang et al. (1993) and Huang and Wagner acts with both TBP and TAF 250, the relative contribution II (1994) were digested with XbaI and Asp718 restriction of binding of different proteins at the initiator element enzymes, and the resulting fragments containing the may account for the quantitative difference in the contri- various VP5 promoter regions and part of the ␤-galacto- bution of the VP16 Inr to full promoter strength compared sidase gene were cloned into a pAlter-1 Vector, from the to that of the VP5 Inr. Thus, defining the exact elements commercial Altered Sites II Mutagenesis Systems (Pro- and proteins binding to these sites is important in un- mega). derstanding the mechanisms of HSV-1 gene regulation. An oligonucleotide containing the mutations of the Sp1 In conclusion, the superficial similarities between the site at Ϫ48, GGGG(TϾa)GG(GGϾtt)CGGGG, along with VP16 and VP5 promoters in the arrangement of Sp1 the ampicillin repair oligonucleotide was annealed to the binding sites are so marked as to suggest a common single-stranded DNA template. The mutant promoters origin, but clearly their functional role in promoter activity were synthesized by T4 DNA polymerase and ligated has not been conserved. While the data presented in this with T4 DNA ligase using procedures described in detail paper describe the situation at a time late after infection in the supplier’s (Promega) technical manual. The result- since the promoter attains maximum activity then, we ing plasmids containing the mutated Sp1 at Ϫ48 were have determined that the specifics of functional architec- digested with XbaI and Asp718 and cloned into the gC ture and their differences are seen at early times also recombination cassette to generate recombinant vi- (data not shown). These differences and the presence of ruses. potentially redundant elements fully underscore the need for complete functional analysis of any HSV promoter Generation of VP16 promoter/leader deletions before it is operationally grouped with others. We have recently shown that the VP16 promoter can functionally We started with a cloned VP16 promoter fragment substitute for the VP5 promoter controlling expression of extending from Ϫ276 to ϩ153 relative to the cap site and the major capsid protein at its wt locus in the viral containing an XbaI linker at the 5Ј end described in Blair genome as assayed by virus replication in cultured cells et al. (1987). This corresponds to bases 105,533 to (Lieu and Wagner, 2000). It will be of interest to measure 105,258 of the 17 synϩ genome (McGeoch, 1991). This the effect of such a substitution on the pathogenesis clone was digested with XbaIatϪ276 and EcoRV at and/or tissue-specific expression of this protein in ani- ϩ153 and subcloned into pUC20. This plasmid was lin- mals. The differences between these promoters, and earized with EcoRV and subjected to digestion with especially the fact that the VP16 promoter contains a Bal31 exonuclease from IBI (Sambrook et al., 1989). Fol- putative tissue-specific transcription factor binding site, lowing digestion, phage T4 DNA polymerase (Boehringer reinforce this interest. Mannheim) was used to blunt DNA ends and HindIII linkers were ligated. Plasmids were ligated together and MATERIALS AND METHODS transformed in Escherichia coli DH5␣. Various leader ϩ ϩ ϩ Cells lengths generated were 95, 32, and 6. These were digested with XbaI and HindIII and subcloned into the Rabbit skin fibroblasts and Vero cells were maintained HindIII site upstream of the reporter bacterial ␤-galacto- at 37°C under 5% carbon dioxide in Eagle’s minimum sidase gene in the gC recombination cassette described essential medium containing 5% cadet calf serum, 100 U elsewhere (Huang et al., 1993; Guzowski and Wagner, of penicillin, and 100 ␮g of streptomycin per milliliter. 1993; Guzowski et al., 1994). Generation of the 5Ј deletions of the VP16 promoter Virus was performed by polymerase chain reaction with spe- The wild-type HSV-1 strain 17 synϩ was used to gen- cific oligonucleotides. The VP16 promoter sequence ex- erate recombinant viruses containing the modified VP5 tending from Ϫ276 to ϩ6 in conjunction with the 5Ј 213 200 LIEU AND WAGNER bases of the ␤-galactosidase reporter gene was sub- to the deletion end-points. The wt cap sequences were cloned into pGEM3 as an XbaI–Asp718 fragment. The built back from Ϫ15 by ligating the double-stranded oli- upstream primers for PCR were single-stranded oligonu- gonucleotides containing sequences from Ϫ15 to ϩ6 cleotides with an XbaI linker and 20 bases of the pro- with the 5Ј AvaI linker and a 3Ј HindIII linker. Similarly, moter sequence starting from Ϫ34, Ϫ46, Ϫ52, Ϫ86, the mutated cap sequences were generated by ligating Ϫ100, and Ϫ136, relative to the transcription start site. the double-stranded oligonucleotides containing the mu- The downstream primer binds in the 5Ј 213 bases of the tated cap sequence Ϫ15 (5Ј-CTCCCCCGGGCTAGT- ␤-galactosidase sequence overlapping the Asp718 site. GCGTCTCCGGGG-3Ј)ϩ6. These constructs were se- This was used to generate deletions by the standard quenced with the SP6 primer. PCR methods: 94°C for 1 min, 55°C for 1 min, 72°C for 1 Construction of mutated TATA and Inr elements utilized min for 45 cycles, followed by a final extension at 72°C the commercially available Quick Change Site-Directed for 10 min. The PCR products were extracted with phe- Mutagenesis Kit from Stratagene. The VP16 promoter nol–chloroform and deposited by ethanol precipitation. containing sequences from Ϫ276 to ϩ6 and the 5Ј region The resuspended material was then digested with XbaI of the ␤-galactosidase gene described above was sub- and Asp718 and separated by agarose gel electrophore- cloned into pGEM3 at the XbaI and Asp718 sites. The sis. The appropriate size fragments were eluted and mutagenic oligonucleotide containing the mutated TATA cloned into the pGEM3 vector. After the ends and se- sequence or both mutated TATA and Inr elements was quences were confirmed, they were cloned back into the annealed to the VP16 promoter. The mutant promoter gC recombination vector as XbaI–Asp718 fragments. was synthesized by PCR with reagents and enzymes from the kit following the detailed methods described in Mutation of VP16 promoter elements the Strategene manual. Mutations were confirmed by sequencing. The sequences of all mutated elements are shown under Results and in the appropriate figure legends. The DNA mobility shift assays Sp1 site near Ϫ46 was mutated as follows. The VP16 promoter sequence extending from Ϫ276 to ϩ6 fused Rabbit skin cells were mock-infected or infected with upstream of the bacterial ␤-galactosidase gene de- HSV-1 at a multiplicity of infection (m.o.i.) of 5 PFU per scribed above was digested with XbaI and Asp718 and cell. After a 1-h adsorption period, infection was allowed subcloned into the p-Altered-1 Vector (Promega), and to proceed for 7 h, and nuclear extracts were prepared mutagenesis was performed using a commercial Altered as described in Huang and Wagner (1994), Guzowski et Sites II Mutagenesis Kit (Promega). A mutagenic oligo- al. (1994), and Lee et al. (1988). Electrophoretic mobility nucleotide containing the mutated Sp1 site at Ϫ46 was shift assays with DNA probes were carried out by stan- annealed to the VP16 promoter, and the mutant promoter dard methods (Chodosh, 1989; Guzowski et al., 1994). was synthesized by T4 DNA polymerase and ligated with Each assay contained 2 ␮g of nuclear extract, 0.2 ng of T4 DNA ligase using procedures described in detail in 32P-labeled DNA fragment, and 2 ␮g of poly(dI:dC) in a the supplier’s (Promega) technical manual. total volume of 10 ␮l. The binding reactions were allowed Construction of the mutated Sp1 sites from Ϫ123 to to proceed for 30 min at room temperature and com- Ϫ133 and the CAAT box at Ϫ77 utilized oligonucleotide- plexes were detected by electrophoresis conducted at directed mutagenesis by PCR. Plasmids containing the 200 V for approximately2hat4°Con4.5% polyacryl- VP16 promoter sequence beginning at Ϫ133 and Ϫ86 amide gels. For competition experiments, the unlabeled were subcloned as XbaI–Asp718 fragments into pGEM3 oligonucleotides were added in a 10- to 250-fold molar vector. Single-stranded oligonucleotides containing the excess of the radiolabeled DNA probe prior to incubation mutated CAAT box at Ϫ77 and mutated Sp1 sites from with nuclear extracts. Ϫ123 to Ϫ133, along with the lower strand of the oligo- Gel shift assays with GST–YY1 fusion protein were nucleotide to the ␤-galactosidade sequence, were used performed with probes containing wt VP16 Inr, mutant to generate mutations by PCR as described in the pre- VP16 Inr, and VP5 Inr described below. Each assay con- ceding section. The PCR products were cloned into the tained 50 ng of GST–YY1 fusion protein (obtained from pGEM3 vector and sequenced to confirm the mutations Dr. T. Osborne), 0.2 ng of 32P-labeled DNA fragment, 50 and to ensure that no new mutations were introduced ng of dIdC, 20% Ficol, 5 ␮lofZЈ buffer (Bennett et al., before cloning back into the gC recombination cassette. 1999), and 1 ␮l of 5 mg/ml nonfat milk incubated for 20 Mutations in the VP16 Inr region were generated as min on wet ice. Following the incubation, 2 ␮l of loading follows. The VP16 promoter containing the sequence dye (0.1% bromphenol blue, 0.1% xylene cyanol) was from Ϫ276 to ϩ6 in conjunction with part of the ␤-galac- added to each sample. The samples were run on a 4.5% tosidase gene was subcloned into pGEM3. This plasmid nondenaturing polyacrylamide gel in 0.22ϫ TBE at 450 V was digested with HindIII at ϩ6 and shortened from ϩ6 at 4°C. The gels were then dried and subjected to auto- to Ϫ15 by using Bal31 deletion. An AvaI linker was added radiography at 70°C with an intensifying screen. LEAKY-LATE HSV-1 PROMOTER ELEMENTS 201

Electrophorectic mobility shift assays were also per- precipitation method (Wagner et al., 1998; Goodart et al., formed with the HeLa nuclear extracts and Sp1 antibody 1992; Bludau and Freese, 1991). (the Nushift Kit, purchased from Geneka Biotechnology, Recombinant viruses were isolated by limiting dilution Inc.) and purified Sp1 extracts obtained from Promega. and screened on both rabbit skin fibroblasts and Vero Assays were carried out with ds oligonucleotides de- cells as described in Guzowski and Wagner (1993). Two scribed below: the VP16 promoter sequences from Ϫ138 independent isolates from separate transfections were to Ϫ111, containing two potential Sp1 binding sites at generated to ensure that both exhibit equivalent levels of Ϫ123 and Ϫ133; mutations at Ϫ123 (m123) or mutations ␤-galactosidase expression. Pure isolates were ana- at Ϫ133 (m133), and oligonucleotides spanning se- lyzed by PCR and the promoter regions were sequenced quences from Ϫ61 to Ϫ33, containing the potential Sp1 using the Sequenase kit (U.S. Biochemical) as described site at Ϫ46. For supershift analysis, 2 ␮l of the Sp1 elsewhere (Huang et al., 1993; Guzowski et al., 1994; antibodies was added to HeLa nuclear extract prior to Guzowski and Wagner, 1993). the addition of labeled probes. Reaction protocols are described in detail in the manuals of the Nushift Kit from RNA isolation and primer extension analysis Geneka Biotechnology, Inc. and the gel shift assay sys- Rabbit skin fibroblasts were infected with recombinant tem from Promega. viruses at a m.o.i. of 5 PFU per cell. Infections were allowed to proceed for 7 h, and total RNA was isolated DNA oligonucleotide probes using the Trizol Reagent (Gibco BRL). Primer extension analysis was carried out with 10 ␮g of total RNA with 10 A ds oligonucleotide containing the VP16 promoter fmol of 32P-labeled primers. A primer extending from 48 from Ϫ15 to ϩ6(5Ј-CTCCCCCGGGCTGTCATTCCTC- bases in the ␤-galactosidase gene was used to detect CGGGG-3Ј) was used as the probe for VP16 DNA mobil- reporter gene expression for the VP5 and VP16 promoter ity shift assays. A ds oligonucleotide extending from Ϫ11 in the gC locus (Huang and Wagner, 1994; Guzowski et to ϩ15 relative to the VP5 cap site was used in the al., 1994). Primers specific for VP16 wt and gD transcripts competition assays. The single-stranded oligonucleo- were used as internal controls, which give rise to the tides were end-labeled with T4 polynucleotide kinase 66-nt band and 85-nt doublet band, respectively. and [␥-32P]ATP and then annealed. The competing ds All primer extension signals were visualized by scan- oligonucleotides used were the mutated VP16 cap se- ning a PhosphoImager screen (Molecular Dynamics) and quences Ϫ15 (5Ј-CTCCCCCGGGCTAGTGCGTCTC- quantified by using IP Lab Gel software (Signal Analysis CGGGG-3Ј)ϩ6, the VP5 Inr Ϫ11 (5Ј-GATCCCCCGGGTCT- Corp.) as described (Lieu et al., 2000). GTTGGGGACACT-3Ј)ϩ15, and the canconical Sp1 site (5Ј-ATTCGATCGGGGCGGGGCGAGC-3Ј). The ds oligo- ACKNOWLEDGMENTS nucleotides for HeLa nuclear shift and Sp1 antibody assays were as follows: sequences from Ϫ61 to Ϫ33 We thank Dr. Tim Osborne and Mary K. Bennett for providing the YY1 (CACGAGGTACGGGGCGGCCGTGCGGGT) of the VP16 protein and protocols for the gel shift assays. M. Rice provided invalu- promoter containing the potential Sp1 binding site at able technical assistance. G. B. Devi-Rao, M. Rice, S. Aguilar, and S. Ϫ Ϫ Stingley provided useful input and critical reviews of the manuscript. 46; mutations at 46 (CACGAGGTACGCTGCAGCCGT- This work was supported in part by Grant CA11861 from the National GCGGGT); sequences from Ϫ138 to Ϫ11 (CCGTGACT- Institutes of Health and by a predoctoral fellowship to P. T. Lieu from the GGGTGGCGGGGGGTGGGTTGGACA) containing two Gene Therapy for Cancer Training Grant of the University of California. potential Sp1 sites at Ϫ123 and Ϫ133; mutations at Ϫ133m (CCGTGACTGCAGTTCTGCGGGTGGGTTGGACA); REFERENCES and mutations at Ϫ123m (CCGTGACTGGGTGGCGGG- Athanikar, J. N., and Osborne, T. F. (1998). Specificity in cholesterol GATCGTATTGGACA). regulation of gene expression by coevolution of sterol regulatory DNA element and its binding protein. Proc. Natl. Acad. Sci. USA 95, Generation and analysis of recombinant viruses using 4935–4940. the gC recombination cassette Bennett, M. K., Ngo, T. T., Athanikar, J. N., Rosenfeld, J. M., and Osborne, T. F. (1999). 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