JOURNAL OF VIROLOGY, Dec. 1991, p. 6705-6713

Vol. 65, No. 12

0022-538X/91/126705-09$02.00/0 Copyright C) 1991, American Society for Microbiology

Sequence, Function, and Regulation of the Vmw65 Gene of Herpes Simplex Virus Type 2 RICHARD F. GREAVESt AND PETER O'HARE*

Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 OTL, United Kingdom Received 10 June 1991/Accepted 21 August 1991

We determined the sequence of the gene for the virion transactivator protein Vmw65 of herpes simplex virus type 2 (HSV-2), strain 333. An analysis of the coding sequence revealed an overall high degree of primary sequence conservation (86%) relative to the HSV-1 protein, although the carboxy-terminal region which encompasses the powerful acidic transactivation domain of the HSV-1 protein was slightly less well conserved (70%). One important change in this region was the presence of a proline residue in a region of the HSV-2 protein which is thought to form an amphipathic alpha-helix in the HSV-1 homolog. Despite the occurrence of this helix-disrupting residue, the HSV-2 protein exhibited powerful transactivation properties for immediateearly target promoters. We also demonstrated that the HSV-2 protein forms a transcriptional complex (TRF.C) with the cellular Oct-i protein and target TAATGARAT elements from immediate-early promoters. A comparison of upstream sequences from the two Vmw65 genes revealed good conservation of proximal promoter elements but considerable divergence elsewhere. Specifically, the HSV-2 promoter alone carries 9.5 copies of a 9-bp direct repeat (GGGGCGGGA) ending 85 bp upstream of the conserved TTAAAT element. An analysis of transcription factor binding sites in vitro revealed that cellular factor Spl bound to the direct repeat sequence of the HSV-2 promoter and that cellular factor USF bound to a proximal element present in both HSV-1 and HSV-2 promoters. Mutational analysis of the HSV-2 promoter demonstrated that the integrity of both of these binding sites was important for the full activity of the promoter.

The virion protein Vmw65 (VP16, a-TIF, ICP25) of herpes simplex virus (HSV) performs two known functions during the virus life cycle. It is a major structural component of the virion (25, 52) and as such has an essential function for normal virus assembly (1, 41). Vmw65 also specifically transactivates immediate-early (IE) gene expression after virus infection (9, 47). This function is dispensible for infection in tissue cultures at a high multiplicity but is essential for normal virus replication at low multiplicities of infection and for virulence after intracranial or intraperitoneal inoculation in mice (2). Although Vmw65 does not independently bind DNA (35, 37), it forms a complex with cellular transcription factor Oct-1, which binds to the octamer or TAATGARAT motifs present in HSV IE genes (37, 42, 43, 48, 49, 53). Induction of transcription is mediated by the acidic transactivation domain located at the carboxy terminus of Vmw65. Mutagenesis of the HSV type 1 (HSV-1) protein has allowed the dissection of these constituent activities (20, 55). Residues 49 to 388 of HSV-1 Vmw65 are sufficient for the formation of transcriptional complex TRF.C (21), which includes Vmw65, a TAATGARAT element, Oct-1 protein, and a second cellular factor (27, 30, 61). The formation of this complex correlates tightly with transactivation by wild-type Vmw65 (21, 43), providing a mechanism for selective location of the activation domain (which is itself dispensible for TRF.C formation) onto target genes. We have demonstrated that point mutations in the region between residues 366 and 387 frequently affect the ability of the Vmw65 protein to participate in TRF.C and have predicted that this region is directly involved at an interface within the TRF.C complex

(21). Consistent with this proposal is the abolition of complex formation by the insertion of short two- to four-residue peptides at (among others) position 369 or 379 (1, 59). Furthermore, we have shown that a peptide derived from this region can specifically interfere with transcriptional complex formation (23). The acidic transactivation domain is contained within residues 413 to 490 of Vmw65 and retains function when linked to the DNA binding domains of other proteins (11, 50). This region is predicted to form amphipathic alphahelices, consistent with the proposal that helix formation is a requirement for acidic activation function (34). Surprisingly, however, the recent results of Cress and Triezenberg (12) indicate that alpha-helix formation may be unimportant and that specific hydrophobic interactions and an overall negative charge are the crucial features required for function. The acidic region is thought to activate transcription by contacting TFIID (54), TFIIB (33), or a transcriptional adapter (4, 28). The Vmw65 protein has two identified homologs, both among the alphaherpesviruses. The Vmw65 protein of HSV-2 has been shown to activate IE gene transcription (45), although its sequence is presently unknown. In contrast, the Vmw65 protein of varicella-zoster virus, UL10, whose sequence has been determined (15), seems not to act as a transcriptional regulator or to form a complex with cellular transcription factors (21a). Knowledge of the sequence and properties of the HSV-2 homolog will clearly be useful in advancing our understanding of structure-function relationships within Vmw65. MATERIALS AND METHODS

Plasmids, mutagenesis, and sequencing. Plasmids pRG90, pRG91, and pRG131 were used for double-stranded sequencing of the HSV-2 Vmw65 gene from strain 333 and

* Corresponding author. t Present address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5402.

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contain, respectively, 1.6-kbp KpnI, 0.75-kbp XhoI-KpnI, and 1.2-kbp SstI-KpnI fragments from plasmid pGR135 (45) inserted into pUC19. Expression vector pRG1 contains HSV-2 strain 333 sequences from an XhoI site in the untranslated leader of Vmw65 to an SstI site in the next open reading frame downstream, cloned via linkers between the HindIII and EcoRI sites of plasmid pCMV-IL2 (13). This procedure places the HSV-2 Vmw65 coding sequence under the control of the powerful human cytomegalovirus IE promoter in a vector which contains the simian virus 40 origin of replication and which can therefore replicate in COS cells. The analogous expression vector pRG70 expresses Vmw65 from HSV-1 strain MP and has already been described (21). Plasmids pRG154, pRG155, pRG163, and pRG164 (see Fig. 7 for a summary) all contain Vmw65 promoter sequences cloned (via linkers) in the appropriate orientations upstream of the chloramphenicol acetyltransferase (CAT) coding sequence of plasmid pCATB' (58). The HSV-1 promoter sequence was numbered by designating the central T of the sequence GCTGT + 1 (see highlighting in Fig. 2). This site has been mapped as the mRNA cap site (+2 bases) by Dalrymple et al. (14) and Pellet et al. (46). The HSV-2 promoter sequence was numbered by naming the homologous T residue in the HSV-2 promoter +1 also (see Fig. 2). Plasmid pRG154, the parent construct for promoter studies, contains a 418-bp XhoI fragment from the Vmw65 promoter of HSV-2 strain 333, stretching from -288 (in the penultimate codon of the HSV-2 UL49 homolog) to + 131, 34 bp upstream of the predicted initiator codon for Vmw65. Mutations were introduced into the promoter sequences by subcloning the sequences into pTZ vectors (39) and performing oligonucleotide-directed mutagenesis on singlestranded derivatives (31) with a Bio-Rad Mutagene kit. All mutations were verified by double-stranded sequencing of plasmids. Plasmid pRG164 is identical to pRG154 except for a single point mutation resulting in an XhoI site at -57. Plasmid pRG174 is identical to pRG154 except for two transversions which generate an SphI site at -109. Plasmid pRG178 is similar to pRG174 except that sequences upstream of the MluI site at -211 have been deleted. Plasmid pRG163 is similar to pRG174 except that the viral sequences upstream of the novel SphI site (-109) have been deleted. See Fig. 7 for a summary of these plasmids. Plasmid pBB5 encodes the IEllOK gene and contains a 4,365-bp BstXI fragment of HSV-1 strain MP cloned into the SmaI site of pUC19. This fragment contains IEllOK promoter and coding sequences but lacks LAT promoter sequences (3). Plasmid pBB37 (2a) encodes the IE175K gene under the control of the human cytomegalovirus IE promoter and contains an approximately 5-kbp SalI-DraI fragment of HSV-1 strain MP cloned into vector pCMV-IL2 (13). Reporter plasmid pAB5 (42) contains the HSV-1 IEllOK promoter cloned upstream of the coding sequence for CAT. Plasmid pPOH3 (44) contains the thymidine kinase promoter of HSV-1 cloned upstream of the coding sequence for CAT. Plasmid sequences were determined by chain termination sequencing with a Sequenase 2 kit (USB). Plasmids were prepared for analysis by alkaline denaturation. For the HSV-2 Vmw65 gene sequence, plasmids pRG1, pRG90, pRG91, and pRG131 were used as templates. For comparative purposes, we also sequenced the promoter region of the Vmw65 gene from HSV-1 strain MP. This sequence was identical to the published sequence (46) for HSV-1 strain F, with the single exception that it lacked a G residue at -68. There are only two additional changes between the se-

J. VIROL.

quences of these two strains and the sequence of HSV-1 strain 17, which contains a T residue instead of a G residue at position -17 and lacks a T residue at position +78 (14). Universal primers were used to commence HSV-2 sequencing; thereafter, 15-bp primers derived from the determined sequence were synthesized and used to extend the sequence until the complete gene was sequenced on both strands. All oligonucleotides were synthesized on an Applied Biosystems 381A synthesizer. Complementary oligonucleotide pairs for gel retardation studies were annealed by being heated together to 70°C and then slowly cooled to 30°C. Oligonucleotide pairs containing binding sites for transcription factors PHO4, USF, AP-1, and Spl were kindly provided by Colin Goding. Gel retardation analysis. Cell extracts were incubated with probes in 20 ,ul of binding buffer, which contained 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.9), 50 mM KCl, 5 mM dithiothreitol, 1 mM sodium EDTA, and 0.05% Nonidet P-40. A 5-min incubation at 20°C with nonspecific DNA (0.5 ,ug of sonicated salmon sperm DNA per ,ul of nuclear extract; 5 ,ug/l, of whole-cell extract) was followed by a 20-min incubation at 20°C with specific unlabelled competitor oligonucleotides (when appropriate) and then by a 30-min incubation at 20°C with approximately 0.5 ng of end-labelled oligonucleotide probe. Gel retardation analysis (18) was performed on 4% nondenaturing polyacrylamide gels with a 19:1 acrylamide/bisacrylamide ratio as described previously (21). Quantitative estimates of labelled probe bound were made by liquid scintillation counting of retarded bands excised from dried

polyacrylamide gels.

Transient expression assays. COS cells and Vero cells were routinely cultured in Dulbecco modified Eagle minimal es-

sential medium containing 10% newborn calf serum. Transfections and CAT assays were carried out in COS or Vero cells as previously described (21). When appropriate, cells were infected at 18 h postinfection with 5 PFU of HSV-1 strain MP per cell and harvested 22 h later. A quantitative estimate of chloramphenicol acetylation was obtained by excision of the substrate and products from thin-layer chromatography plates and subsequent measurement by liquid scintillation counting in an LKB 216 scintillation counter. Activity is expressed as counts per minute appearing in the

acetylated products. Cell extract preparation and protein analysis. For largescale nuclear extract preparation, HeLa cells (1 x 1010 to 2 x 10l° cells) were grown in suspension in Joklik modified Eagle minimal essential medium containing 10% newborn

calf serum. Nuclear extracts were prepared by the method of et al. (16). For preparation of transfected-cell extracts, 2.5 x 107 COS cells were transfected with 20 ,ug of expression vector by the method of Chen and Okayama (10) with N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acidbuffered saline. Whole-cell extracts were prepared after 40 h as described by Wu (60). For the comparison of transactivation and complex formation in cells transfected with Vmw65 from HSV-1 or HSV-2, equal aliquots of both total cellular material and solubilized extract were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (32) followed by Western immunoblotting (7), using monoclonal antibody LP1 (38) as previously described (20, 21).

Dignam

Nucleotide sequence accession number. The 2,228-bp sequence determined in this work was submitted to the GenBank data base and can be retrieved under accession number M75098.

VOL. 65, 1991

HSV-2 Vmw65

1- MDLLVDELFADMNADGASPPPPRPAGGPKNTPAAPPLYATGRLSQAQLMP HSV-1

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11111 11 1 111111 III 11 1 -188 AGGGGCGGGAGGGGCGGGAGGGGCGGGAGGGGCGGGAGGGGCGGGAGGGG 9.5 perfect direct 9bp repeats - SP1 sites

11:: ll::Iliii 111111 11111 111111111111111111111111

149- EAMAQFFRGELRAREESYRTVLANFCSALYRYLRASVRQLHRQAHMRGRD 201- RDLGEMLRATIADRYYRETARLARVLFLHLYLFLTREILWAAYAEQMMRP

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III 11 11 III 11 I 11 11 -138 CGGGAGGGGCGGGAGGGGCGGGAG=GGCGACACGCCTCCCTTCCGAGCGC

111 1111 11111 11111111111111111111:11 111111 1111111

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CTF? USF SPi? -GTG-GGGGAAGTCACGAGGTACGGGGCGGCC 1111 1I 1111111111111111 11111111 -88 GGGGGACGGGCCGCCCGGAGCGTGGGGAAGTCACGAGGTTTGGGGCGGCA USF SPi?

USF?

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251- DLFDCLCCDLESWRQLAGLFQPFMFVNGALTVRGVPIEARRLRELNHIRE 1111 111111111111l 1111:11:11:1111111:1111111111111 249- DLFDGLCCDLESWRQLACLFQPLMFINGSLTVRGVPVEARRLRELNHIRE 301- HLNLPLVRSAATEEPGAPLTTPPTLHGNQARASGYFMVLIRAKLDSYSSF

299- HLNLPLVRSAAAEEPGAPLTTPPVLQGNQARSSGYFMLLIRAKLDSYSSV

mRNA cap 'TATA'box -38 CGTGCGGGTTGCTTAAATGCGGGGTGGCGACCACGGGCT?TCATTCCTCGl

-38 CTGGGGTGATACTTAAATGCGGGGTGGTGGACGCGAGATGTCAGTCCTCG 'TATA'box

region required for TRF.C formation 351- TTSPSEAVMREHAYS T1fl~tIZb~?D1~D+APEEAGLAAPRL

+13 GGAACGGACGGGGTTCCCGCTGCCCACTTCCCCCCATAAGGTCCGTCCGG III 11111 111111111 I111 1111 +13 GGGACGCACGGCACCCCCGGCGA----TTCCCTTCGCGAGGGCC--CCGG

349- ATSEGESVMREHAYSRGRTRNNYGSTIEGLLDLPDDDDAPAEAGLVAPRM

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FIG. 1. Deduced primary sequence of the HSV-2 strain 333 Vmw65 protein, aligned with the primary sequence of the Vmw65 protein from HSV-1 strain 17 (14). Identical residue pairs are indicated by a vertical line, and semiconserved residue pairs are indicated by a colon. Residues in the HSV-1 protein required for TRF.C formation are shaded, as is the proline residue at position 436 in the HSV-2 activation domain.

RESULTS HSV-2 Vmw65 protein sequence. We obtained the sequence (Fig. 1 and Fig. 2) of 2,228 bp of the HSV-2 strain 333 genome known to contain the gene for Vmw65 (UL48 in the nomenclature of McGeoch et al. [36] for HSV-1). Our determined sequence begins with the last 10 codons of the presumptive HSV-2 UL49 homolog. The stop codon for the UL49 reading frame is followed after 29 bp by a consensus polyadenylation signal (AATAAA) (Fig. 2). On the basis of the overall alignment of the HSV-2 sequence with the corresponding sequence of HSV-1 (14, 46) and the assumption that the primary sequence homologies, including, for example, the TATA box sequence, are indicative of transcript homology, the cap site and initiator codon of the HSV-2 Vmw65 gene can be predicted with virtual certainty (Fig. 2), particularly since the resultant mRNA would have the capacity to encode a 490-residue protein with 86% primary homology to the HSV-1 Vmw65 protein (Fig. 1). The end of the HSV-2 open reading frame is followed after 91 bp by a consensus polyadenylation signal (AATAAA) and subsequently by a TATAA signal, which may be the TATA box site of the HSV-2 UL47 homolog. The complete DNA sequence of the region can be retrieved from the GenBank data base under accession number M75098.

EcoRV

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FIG. 2. Alignment of nucleotide sequences upstream of the reading frames for Vmw65 in HSV-1 strain MP and HSV-2 strain 333. The stop codons and polyadenylation signals for UL49 are shaded, as are the initiator codons for Vmw65. Restriction sites used for reporter constructs are marked, and the positions of point mutations used to create SphI sites are indicated by asterisks. Also shaded are the putative TATA boxes, the HSV-1 transcription start site (14, 46), the binding site for USF demonstrated in this paper, and the Spl binding direct repeats of the HSV-2 promoter.

There is very good alignment between the HSV-1 and HSV-2 proteins (86% homology), and the greatest divergence in the primary sequence occurs in the carboxylterminal acidic domain (70% homology). The overall strong homology is consistent with conservation of the dual roles of Vmw65 as an essential structural component and as the virion transactivator of IE genes. As for most regions of the genome, the region encoding Vmw65 can be contained within viable intertypic recombinants (40). The primary sequence of the HSV-2 protein in the region from residues 366 to 388 (which we have demonstrated to be crucial for Vmw65-Oct-1 complex formation) showed two fairly conservative changes (Ala-367 to Gly; Lys-370 to Arg) (Fig. 1). Although we have determined that lysine 370 is critical for complex formation in the HSV-1 protein, we have also shown that its mutation to arginine is tolerated, consistent with the presence of arginine at this position in the HSV-2 protein (21b). An analysis of the primary sequence of the acidic domain of the HSV-2 protein revealed a more unexpected feature: the presence of a proline residue at position 436 (Fig. 1). The

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FIG. 3. (a) Transactivation of the IE 110K-CAT construct by cotransfection with Vmw65 expression vectors. COS cells were transfected with 20 ng of target plasmid and 10 ng of pRG50 for HSV-1 Vmw65 expression, pRG1 for HSV-2 Vmw65 expression, or control vector pCMV19A for basal activity. CAT activity in soluble extracts

was

measured at 40 h posttransfection. Ac-CAMP, acety-

lated chloramphenicol products; CAMP, chloramphenicol substrate. (b) Formation of the TRF.C transcriptional complex on a TAATGARAT element by HSV-1 Vmw65 and HSV-2 Vmw65. End-labelled oligonucleotide TAAT24 (1 ng) was incubated together with a HeLa cell nuclear extract (1 ,ul) and extracts of transfected COS cells (1 ,ul). Bound oligonucleotide was resolved by nondenaturing electrophoresis; unbound probe is not shown in this figure. Vmw65 was expressed in COS cells by transfection with vector pRG50 (HSV-1) or pRG1 (HSV-2). The binding profile of an extract made from cells transfected with the control vector is shown in parallel (pCMV19A). Approximately equal amounts of Vmw65 were detected in pRG1- and PRG50-transfected cells by Western blot analysis of the extracts with type-common monoclonal antibody LP1 (data not shown).

acidic activation domain has been predicted to form two amphipathic alpha-helices, but the presence of a proline residue in the HSV-2 protein may preclude the formation of at least one of these structures. Alternatively, if a helix does form in this region, it would be restricted to around residues 438 to 448. This region of the HSV-2 protein is clearly functional in transcriptional activation, as we show below; we and others have also demonstrated that an Ala-to-Pro switch at an adjacent residue in the HSV-1 activation domain has no detectable effects on its function (12, 41a). Despite the slightly lower level of sequence homology between the acidic domains, key features are clearly well conserved. Negative charge and its distribution are very similar in the two proteins (all acidic residues present in the HSV-1 activation domain are present in its HSV-2 equivalent), although there are three Asp-to-Glu changes. The carboxylterminal region of the HSV-2 protein also has two additional acidic residues as the result of Ala-to-Asp changes. HSV-2 Vmw65 protein function. An expression vector (pRG1) which expresses Vmw65 of HSV-2 strain 333 was constructed. The vector used has a simian virus 40 origin of replication and the IE promoter from human cytomegalovirus and can be used to produce large quantities of foreign protein in transfected COS cells. We previously described similar vectors which express the Vmw65 protein of HSV-1 strain MP (20). Assays for transactivation of an IE promoterCAT construct by HSV-1 and HSV-2 proteins are shown in

Fig. 3a. Cotransfection of vectors expressing Vmw65 from HSV-1 or HSV-2 stimulated CAT expression to similar extents, indicating that the proline residue at position 436 in the HSV-2 acidic domain has no substantial effect on the activation function. Figure 3b shows that HSV-2 Vmw65 overexpressed in COS cells is also fully functional for the formation of transcriptional complex TRF.C with cellular proteins and target promoter sequences. This result was as we would predict, since TRF.C formation seems essential for transactivation (1, 21, 43, 59). The HSV-2 Vmw65 promoter contains a repeat structure. The intergenic region between the UL49 and Vmw65 (UL48) reading frames from HSV-1 strain MP and HSV-2 strain 333 is shown in Fig. 2. The homology within this region is clearly not as great as that within the protein coding region. However, there is very clear homology just upstream of the mapped cap site of the HSV-1 transcript. This homology includes a conserved CTTAAATGCG sequence at -27, presumably the functional TATA box (14, 46), a GGGGCG GC sequence at -47, and a GGGGAAGTCACGAGGT sequence at -65. On the basis of the conservation of the nucleotide sequence and spacing, it would seem likely that the latter two elements may represent cis-acting sites shared between the two promoters and may be binding sites for transcription factors. Upstream of the -65 element and downstream of the UL49 polyadenylation signal there is considerably less homology between the two promoters. The HSV-1 promoter carries only unique sequence in this region which contains potential binding sites for transcription factors USF (CACGTG) at -87 and CTF (CCAAT) at -74. In contrast, the HSV-2 promoter has 9.5 copies of a 9-bp direct repeat (GGGGCGGGA) flanked by a unique sequence. These repeated elements closely resemble the binding site consensus sequence for cellular transcription factor Spl (6). On the basis of these observations, it seems that the two promoters share similar structures proximal to the transcription start site but may have different distal elements. Expression and transactivation of the HSV-2 Vmw65 promoter. We constructed plasmids which linked the promoter region to the coding sequence for CAT. The fragment used spanned from the XhoI site at position -288 in the penultimate codon of UL49 to the XhoI site at position +131 in the 5'-untranslated leader region of the Vmw65 gene. The activities of the promoter and mutant versions were assessed by determining CAT activity in transfected Vero cells. A measure of basal activity in a dose-response experiment is shown in Fig. 4a. The HSV-2 promoter construct (pRG154) showed good basal activity, approximately twice that of the delayed-early thymidine kinase promoter construct (pPOH3). The results of virus superinfection of transfected cells are shown in Fig. 4b. The HSV-2 promoter was clearly induced by virus superinfection; the induction ratio relative to mock-infected controls was in the range of 4- to 12-fold. Superinfection by HSV-1 or HSV-2 induced promoter activity to similar levels (data not shown). HSV-1 IE transactivator proteins were introduced separately in cotransfection experiments, and typical results from these experiments are shown in Fig. 4b. Cotransfection with a vector expressing the IEllOK protein induced expression from the HSV-2 promoter by 25-fold, and cotransfection with a vector expressing the IE175K (ICP4) protein induced expression by approximately 15-fold. A conserved binding site for cellular transcription factor USF is required for full activity of the HSV-2 Vmw65 promoter in transient expression assays. The 16-bp sequence GGGGAAGTCACGAGGT is located approximately 65 bp

VOL. 65, 1991

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FIG. 4. (a) Dose-response assays of CAT expression from HSV Vmw65 promoter-CAT constructs. Vero cells (106 cells) were transfected with various amounts of the indicated plasmids, and CAT activity in soluble cell extracts was measured at 40 h posttransfection. (b) Induction of expression from Vmw65 promoter-CAT constructs by superinfection with HSV-1 strain MP or by cotransfection with genes for HSV IE proteins. Vero cells were transfected with pRG154 (1 ,ug) and infected at 18 h after transfection with 5 PFU of HSV-1 per cell. CAT activity in soluble extracts was measured at 20 h postinfection. For the cotransfection experiments, target construct pRG154 (1 ,ug) was transfected into Vero cells together with IEllOK expression vector pBB5 (50 ng) or IE175K expression vector pBB37 (20 ng). CAT activity in soluble cell extracts was measured at 40 h posttransfection. inf, infected.

upstream of the transcription start site for the HSV-1 promoter and is perfectly conserved in both HSV-1 and HSV-2 promoters. To test whether this sequence was a transcription factor binding site, we synthesized 29-bp oligonucleotides spanning the conserved region and examined the protein binding profile in gel retardation assays. Using a nuclear extract of HeLa cells (16) as a source of transcription factors and oligonucleotides from either the HSV-1 or the HSV-2 promoter, we observed identical complexes consisting of a single major species and a minor, faster-migrating species (Fig. Sa). We noted similarities within the probes to the binding sites for cellular factors AP-1 and USF and binding experiments were therefore conducted with unlabelled competing oligonucleotide pairs containing known binding sites for AP-1, USF, Spl, and yeast factor PHO4. Of these, the USF site competed strongly for the specific complex, the PH04 site competed weakly, and the AP-1 and Spl sites did not compete significantly at any of the doses tested (data not shown). We therefore tentatively assigned the binding activity from HeLa cell nuclei as USF. Weak competition by the PH04 site is consistent with this conclu-

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FIG. 5. (a) Binding of HeLa cell nuclear factor USF to similar elements in AdMLP (Ad MLP), the HSV-2 Vmw65 promoter (HSV2), the varicella-zoster virus UL10 promoter (VZV), and the HSV-1 Vmw65 promoter (HSV1). Bound end-labelled oligonucleotide probes were resolved by nondenaturing gel electrophoresis. For purposes of increased resolution, unbound probe was electrophoresed off the gel but was shown to be in excess in parallel experiments. Lanes: -, competition by nonspecific competitor DNA only; c, specific competition by AdMLP USF site oligonucleotides (100-fold molar excess); h, formation of the complex by a heat-treated HeLa cell nuclear extract. (b) Binding of a HeLa cell nuclear factor to USF sites in AdMLP (Ad MLP), the HSV-2 Vmw65 promoter (HSV2), and a mutant HSV-2 Vmw65 promoter (CTCGAG). Bound end-labelled oligonucleotide probes were resolved by nondenaturing gel electrophoresis; unbound probe is not shown. Lanes: 1, competition by nonspecific competitor DNA at 100 ng; 2, competition by nonspecific competitor DNA at 500 ng; 3, competition by nonspecific competitor DNA at 500 ng and AdMLP oligonucleotides (100-fold molar excess). USF, USF complex; n.s., nonspecific complex. (c) Induction of expression from wild-type and mutant Vmw65 promoter-CAT constructs by viral IE proteins supplied by cotransfection. Target promoters (1 ,ug) were transfected into 106 Vero cells together with IEllOK expression vector pBB5 (50 ng) or IE175K expression vector pBB37 (20 ng). CAT activity in soluble cell extracts was measured at 40 h posttransfection. w/t, wild type.

sion, as the PH04 protein is, like USF, a helix-loop-helix protein and binds to a related motif (18a). To confirm our conclusion, we end labelled an oligonucleotide pair containing the USF site from the adenovirus 2 major late promoter (AdMLP) and used it in parallel gel retardation assays. The

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GREAVES AND O'HARE

result (Fig. Sa) shows that the mobilities of complexes formed on the AdMLP USF probe and the HSV element probes were identical (lanes marked -) and that all of the complexes were specifically competed for by the unlabelled AdMLP USF site (lanes marked c). Furthermore, in reciprocal cross-competition experiments, the HSV-2 site competed effectively for USF binding to the AdMLP site. In addition, lanes marked h show that the binding activity from HeLa cell nuclear extracts was relatively resistant to heat treatment (10 min at 80°C), a characteristic of the USF protein (51). We proposed that USF was probably able to bind to the sequence CACGAG in the HSV promoters; this sequence contains a single mismatch from the binding consensus sequence (CACGTG). To confirm this proposition, we synthesized a short oligonucleotide containing only 16 bases around the proposed HSV-2 motif and a corresponding oligonucleotide containing a single base-pair mutation within the site (CACGAG to CTCGAG). The results (Fig. Sb) demonstrated that the short wild-type oligonucleotide bound the factor as efficiently as the longer probe and with an efficiency comparable to that of the AdMLP probe (compare lanes 1 and 2 from the AdMLP panel with the corresponding lanes from the HSV-2 panel). The single point mutation in the HSV-2 sequence virtually abolished binding (Fig. Sb) and, when assayed by competition assays (data not shown), the affinity of USF for this site was apparently reduced by at least 100-fold. To determine the significance of the USF site, we introduced this point mutation into the HSV-2 promoter-CAT plasmid and measured activity in transient expression assays. Typical results are shown in Fig. Sc. The construct containing the point mutation exhibited basal activity approximately three- to fourfold lower than that of the wildtype promoter but remained inducible by the IEllOK and IE175K proteins in cotransfection experiments (Fig. Sc). Over the course of several experiments, the ratio of induction by either the IEllOK or the IE175K protein was approximately the same for the wild-type and mutant promoters. These results suggest that USF binding contributes to the high basal and induced activities of the HSV-2 promoter in transient assays but that it is not required for the mediation of induction by viral IE products. We anticipate that the homologous site is involved in the previously demonstrated constitutive and induced expression of the HSV-1 promoter (5). The 9-bp repeat elements can bind cellular factor Spl and are required for full activity of the HSV-2 promoter in transient assays. The major difference between the HSV-2 and HSV-1 promoters is the presence of the 9-bp repeat element, and we therefore investigated whether the element bound a cellular transcription factor and whether this binding was involved in the HSV-2 promoter activity. A 26-bp end-labelled probe containing two tandem copies of the repeat was generated and was used together with a HeLa cell nuclear extract in gel retardation assays. The probe bound specifically to a factor in the nuclear extract (Fig. 6a). Competition experiments with unlabelled oligonucleotide pairs containing AP-1, USF, yeast PHO4, and Spl sites established that the binding activity seen probably represented cellular transcription factor Spl. An end-labelled probe containing the Spl site of the adenovirus EIla late promoter (19) was run in a parallel gel retardation assay and bound a complex virtually identical in appearance and mobility to that found with the HSV-2 repeat probe (Fig. 6a). Complexes formed with both probes were also effectively

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FIG. 6. (a) Binding of HeLa cell nuclear factor Spl to similar elements in the adenovirus Ella late promoter and the HSV-2 Vmw65 promoter direct repeats. Bound end-labelled oligonucleotide probes were resolved by nondenaturing gel electrophoresis; unbound probe is not shown. Adenovirus EIIa late promoter Spl site oligonucleotides were used as specific competitors in the amounts indicated. (b) Competition for Spl binding to an endlabelled EIla late promoter Spl site probe. Adenovirus Ella late promoter Spl site oligonucleotides and unlabelled HSV-2 repeat oligonucleotides were used as specific competitors in the amounts indicated. Bound end-labelled oligonucleotide probes were resolved by nondenaturing gel electrophoresis; unbound probe is not shown. Spl, Spl complex; n.s., nonspecific complex. (c) Induction of expression from wild-type and mutant Vmw65 promoter-CAT constructs by viral IE proteins supplied by cotransfection. Target promoters (1 pLg) were transfected into Vero cells together with IEllOK expression vector pBB5 (50 ng) or IE175K expression vector pBB37 (20 ng). CAT activity in soluble cell extracts was measured at 40 h posttransfection. w/t, wild type.

competed for by a 100-fold molar excess of unlabelled Ella late promoter Spl site (Fig. 6a). Figure 6b shows a compe-

tition assay to examine the relative affinities of the Ella late promoter Spl site and the two tandem 9-bp repeats for Spl. Unlabelled oligonucleotides at different concentrations were used to compete for Spl binding to a labelled Ella late promoter probe. Quantitation of this experiment by band excision and scintillation counting showed that the affinity of

VOL. 65, 1991

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6711

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Spl for the HSV-2 site was approximately 10- to 20-fold lower than its affinity for the EIla late promoter Spl site. To investigate whether the 9-bp repeats were involved in the activity of the HSV-2 promoter, we introduced an SphI site immediately downstream of the repeats by mutagenesis. Near wild-type levels of expression were seen from plasmids into which this SphI site was introduced (pRG174) or from which unique sequences 5' to the repeats were deleted (pRG178) (Fig. 7). A plasmid (pRG163) in which repeat sequences 5' to the introduced SphI site were deleted was made. Typical results of the effects of this deletion on expression from the HSV-2 promoter are shown in Fig. 6c. Basal expression was reduced by four- to fivefold. However, expression remained open to transactivation by the IEllOK and IE175K proteins, suggesting that the repeats are not required to mediate either of these activities (Fig. 6c). DISCUSSION We have reported the

sequence

of the coding and regula-

tory regions of the Vmw65 gene of HSV-2. A comparison of

the encoded protein sequence with the HSV-1 gene reveals products of identical lengths: 490 residues. Vmw65 peptide sequence conservation between the two viruses (86%) is high and comparable to that seen with other essential HSV proteins, DNA polymerase (90%), alkaline exonuclease (80%), glycoprotein D (82%), and glycoprotein B (86%) (8, 17, 56, 57). The strong sequence conservation seen is consistent with the dual role of Vmw65 in both viruses as a major and essential structural component and as the virion transactivator of IE gene transcription. Our aim at the outset of this work was a comparison of peptide sequences in regions of the proteins required for transactivation. The region between residues 366 and 388, which we have proposed to form an interactive interface during transcriptional complex formation (21, 23), as expected from the overall conservation, showed no major difference between the two viruses. A change in the acidic activation domain was of greater interest. The HSV-2 protein has a proline residue at position 436. This helix breaker is positioned centrally in a region predicted to form a long amphipathic alpha-helix in the HSV-1 protein. If the structure of this region is conserved between the two viruses, the predicted helix may not be formed in either protein. Our observation (41a) that an alanine-to-proline switch at residue 436 does not affect the function of the HSV-1 activation domain would seem to confirm this idea. Our data are consistent with a recent report which demonstrated that proline substitutions at position 432 or 436 had no significant

effect on the transactivation function of Vmw65 (12). Overall, the results suggest either that alpha-helix formation is not necessary for the activity of this region or that if a helix does form, it is a short helix located within the region from positions 438 to 448 (at residue 448, the helix probability significantly declines and the protein sequence enters a proline- and glycine-rich segment). The conservation of the sequence between the two Vmw65 promoters is not extensive and centers around two elements upstream of a conserved TTAAAT element. One of the two conserved elements in the HSV-1 and HSV-2 promoters can bind in vitro to cellular protein USF. USF was characterized as the cellular transcription factor which binds to the hexamer sequence CACGTG in AdMLP (51). It is a member, along with Myo D and c-myc, of the helix-loophelix family of DNA binding proteins (22). The core binding site in the HSV promoters (CACGAG) has a single base-pair mismatch relative to the characterized core site but still binds USF with a high affinity-about 30% that of the AdMLP site. In addition, our results with a USF binding site point mutant in transient assays show that USF binding correlates with efficient expression from the HSV-2 Vmw65 promoter. We have also demonstrated the binding of USF to an element in a conserved position upstream of the predicted TATA box of the varicella-zoster virus ORF10 gene, the homolog of the HSV Vmw65 gene. It is intriguing to speculate that the preservation of this site may reflect some common feature in the regulation of these homologous genes during the virus life cycle. We have searched the HSV-1 genome for potential USF sites in the upstream regions of other genes. The searches revealed possible USF binding sites in the regulatory regions of the UL5, UL33, and UL55 genes. A binding site for USF has also recently been characterized in an early gene promoter of another herpesvirus, human cytomegalovirus (29). The repeated 9-bp motif structure is unique to the HSV-2 promoter. We show that this element is an SP1 binding site and is also required for efficient expression from the HSV-2 promoter. However, neither the USF site nor the Spl site accounts for the inducibility of the promoter by IE transactivators. Some other feature of the proximal promoter or leader sequences must be responsible. It is possible that the difference in promoter structure is reflected in a difference in the temporal regulation of the HSV-2 gene compared with the HSV-1 gene. Previous results demonstrated that CAT constructs containing the promoter for the HSV-1 gene were inducible by virus superinfection and that the kinetics of Vmw65 expression during HSV-1 infection followed classical leaky-late (or

6712

GREAVES AND O'HARE

P-y)-type kinetics (5, 62). Earlier results indicated that Vmw65 expression in HSV-1-infected cells was repressed but was still detected in the absence of virus DNA replication (24, 26). Our current work indicates that the expression of the HSV-2 Vmw65 gene may be unaffected by the inhibition of DNA synthesis (21b), indicating that for HSV-2, Vmw65 expression may follow classical delayed-early (or 3)-type kinetics. Comparative analyses of these two promoters and of the regulation of the expression of Vmw65 in HSV-1- and HSV-2-infected cells are now in progress to examine this point. ACKNOWLEDGMENTS This work was funded by the Marie Curie Memorial Foundation. We thank Tony Minson for continued supply of monoclonal antibody LP1 and Colin Goding for oligonucleotides. The typing skills of Jean Marr are gratefully appreciated.

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