JOURNAL OF VIROLOGY, May 1991, p. 2601-2611 0022-538X/91/052601-11$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 65, No. 5

Elements in the Transcriptional Regulatory Region Flanking Herpes Simplex Virus Type 1 oriS Stimulate Origin Function SCOTT W. WONG AND PRISCILLA A. SCHAFFER* Laboratory of Tumor Virus Genetics, Dana-Farber Cancer Institute, and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 Received 12 December 1990/Accepted 7 February 1991

Like other DNA-containing viruses, the three origins of herpes simplex virus type 1 (HSV-1) DNA replication flanked by sequences containing transcriptional regulatory elements. In a transient plasmid replication assay, deletion of sequences comprising the transcriptional regulatory elements of ICP4 and ICP22/47, which flank oriS, resulted in a greater than 80-fold decrease in origin function compared with a plasmid, pOS-822, which retains these sequences. In an effort to identify specific cis-acting elements responsible for this effect, we conducted systematic deletion analysis of the flanking region with plasmid pOS-822 and tested the resulting mutant plasmids for origin function. Stimulation by cis-acting elements was shown to be both distance and orientation dependent, as changes in either parameter resulted in a decrease in oriS function. Additional evidence for the stimulatory effect of flanking sequences on origin function was demonstrated by replacement of these sequences with the cytomegalovirus immediate-early promoter, resulting in nearly wild-type levels of oriS function. In competition experiments, cotransfection of cells with the test plasmid, pOS-822, and increasing molar concentrations of a competitor plasmid which contained the ICP4 and ICP22/47 transcriptional regulatory regions but lacked core origin sequences resulted in a significant reduction in the replication efficiency of pOS-822, demonstrating that factors which bind specifically to the oriS-flanking sequences are likely involved as auxiliary proteins in oriS function. Together, these studies demonstrate that trans-acting factors and the sites to which they bind play a critical role in the efficiency of HSV-1 DNA replication from oriS in transient-replication assays. are

Initiation of herpes simplex virus type 1 (HSV-1) DNA replication begins at discrete sequence elements or origins of DNA synthesis and requires the expression of at least seven viral genes (Fig. 1A) (55). The HSV-1 genome contains three origins of DNA synthesis: a single copy of oriL, located within the unique long segment of the genome (51), and two copies of oriS, located in the inverted repeats flanking the unique short segment (Fig. 1A) (49). oriL is not essential for virus growth, since deletion mutants lacking a functional copy of oriL have been isolated and are capable of nearly wild-type levels of replication (39). At least one functional copy of oriS appears to be required, since attempts to construct viral mutants lacking both copies of oriS have been unsuccessful (7). Comparison of the DNA sequences of oriL and oriS has revealed similar structural organization and extensive sequence homology (51). oriL is contained within a perfect 144-bp palindrome, while oriS is located within an imperfect palindrome of 45 bp (49, 51). Both origins can support plasmid replication in transfected cells when essential replication proteins are provided in trans by superinfection with HSV-1. Further characterization of the HSV-1 origins has focused on oriS rather than oriL because the large oriL palindrome is deletion-prone upon cloning in bacteria (39, 51). The core origin of HSV-1 oriS consists of a 67- to 90-bp sequence that contains an imperfect 45-bp palindrome (Fig. 2) (11, 31, 49). Included within the palindrome are two binding sites for the origin-binding protein, the product of the UL9 gene, that flank a central 18-bp A+T-rich region (37). It has been postulated that short inverted repeats are *

capable of forming cruciform structures in DNA, suggesting that the palindrome in oriS may exist functionally in this form. Further delineation of the structural requirement for oriS was provided by Deb and Doelberg, who used deletion analysis to demonstrate that the arm of the palindrome adjacent to the short flanking sequence is not required for DNA replication (11). This observation, together with the properties of a series of dinucleotide insertion mutations in the center of the A+T-rich region that maintained the integrity of the palindrome and produced an oscillating effect on origin function, argue that the origin does not exist in the expected cruciform structure as first predicted, but exists in a linear configuration (31). The role of sequences in maintaining oriS in a functional configuration is not known. Enhancement of origin function by transcriptional regulatory elements is a common phenomenon among DNA viruses. First observed in polyomavirus (17), this phenomenon has also been shown for simian virus 40 (SV40) (8, 15, 24, 26, 28, 30), bovine papillomavirus (32, 48), adenovirus (45, 53), and Epstein-Barr virus (23, 44, 56). Thus, for example, the core origin of SV40 DNA replication consists of a 65-bp segment that contains numerous binding sites for large T antigen and a 17-bp A+T-rich region. The core origin has been demonstrated to be sufficient to support initiation of DNA replication both in vivo and in vitro. Replication efficiency in vivo, however, has been shown to be substantially increased by sequence elements which flank the core origin (8, 15, 24, 26, 28, 30, 36). These sequences include GC boxes as well as the transcriptional activator elements of the SV40 enhancer. Based on findings from these systems, DePamphilis (16) has suggested that eukaryotic origins of DNA replication contain two primary components: a core component required specifically for the initiation of DNA replication and

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FIG. 1. HSV genome. (A) Beneath the scale of map units is shown a diagram of the HSV-1 genome, indicating the locations of reiterated sequences (a, b, and c) and their inverted counterparts (a', b', and c') as well as the positions of the three origins of DNA replication (oriL and two copies of oriS). Also shown are the locations of the seven genes that specify proteins required for HSV-1 DNA replication, UL 5, 8, 9, 29, 30, 42, and 52 (55). (B) Diagram of the 822-bp BamHI fragment from plasmid pIE3CAT, containing the intergenic region between ICP4 and ICP22/47. The arrows denote the transcriptional start sites for ICP4 and ICP22/47; the box represents the core origin of oriS. Restriction sites: Ba, BamHI; S, SmaI; Bs, BssHII; H, HpaIl; and N, Narl.

is absolutely essential for DNA replication, while it appears to be dispensable in others (16). Although the precise manner by which DNA replication is activated by transcription factors is not known, several hypotheses have been postulated. Recent studies by Cheng and Kelly support the hypothesis that transcription factors inhibit nucleosome formation throughout the SV40 origin and promoter regulatory region (9). In cell-free origin-function assays in which SV40 origin-containing plasmids were associated with nucleosomes and origin function was diminished, these authors reported that the introduction of a unique nuclear factor I (NF-I) binding site adjacent to the core origin enabled the origin to replicate as efficiently as the wild-type origin. These experiments, the authors suggest, are consistent with the concept that transcription factors generate a nucleosomefree region, making DNA more accessible to replication machinery. In the HSV-1 genome, the origins of DNA replication are located in contexts similar to those of other DNA viruses. oriL is positioned between the divergent transcriptional start sites of the genes encoding the major DNA-binding protein ICP8 and the DNA polymerase (39, 51). The transcriptional regulatory elements surrounding oriL are not well defined. oriS, like oriL, is also located between divergently transcribed genes. These genes encode the immediate-early proteins ICP4 and ICP22/47 (49) (Fig. 1 and 2). The transcriptional regulatory elements within which oriS resides are well characterized and collectively exhibit the properties of an enhancer element (40-42). (Fig. 2A and B). A variety of recognized transcription factors have been reported to bind specifically to oriS-flanking sequences. These include the potent HSV-1 transactivator VP16 (4) and cellular transcrip-

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FIG. 2. Intergenic region between immediate-early genes ICP4 and ICP22/47. (A) Beneath the scale (in base pairs) is shown the 822-bp BamHI fragment containing the intergenic region between ICP4 and ICP22/47. The diagram shows the locations of the transcriptional start sites for ICP4 and ICP22/47 (arrows), as well the binding sites for several recognized transcriptional regulatory elements. TAATGARAT motifs that bind VP16 are shown as hatched boxes; Spl binding sites are shown as stippled ovals; TATA boxes are shown as black rectangles; the GCGGAAC motif is shown as an open box; an NF-III-binding site is shown as a checkered box; CCAAT box-like sequences are shown as open circles; and the core origin, oriS, is shown as a large rectangular box. (B) Nucleotide sequence of the 822-bp fragment shown in panel A. Again, the locations of the transcriptional start sites are shown as arrows. The TAATGARAT motifs, Spl binding sites, TATA boxes, GCGGAAC motifs, NF-III-binding site, and CCAAT box-like sequences are boxed, underlined by dashed lines, underlined by bold lines, boxed with dashed lines, underlined by double lines, and enclosed with rounded boxes, respectively. The two recognized UL9 binding sites are denoted by dark lines, and the oriS palindrome is shown as convergent arrows.

tion factors Spl, nuclear factor III (NF-III) (1), and the factor recognizing the GCGGAAC sequence shown by Triezenberg et al. to be required for efficient expression of ICP4 (50). Collectively, the transcriptional regulatory elements located on both the right and left sides of the core origin promote transcription from both the ICP4 and ICP22/47 promoters early in infection, resulting in the synthesis of three of the five HSV-1 regulatory proteins (40, 47). ICP4 is essential for virus replication and acts to repress transcription of immediate-early genes and activate transcription of early and late genes (13, 14). The roles of ICP22 and ICP47, both immediate-early proteins, have not yet been determined; however, neither is essential for virus replication in cell culture (47).

STIMULATION OF oriS FUNCTION IN HSV-1

VOL. 65, 1991

Early studies designed to identify the cis-acting elements involved in oriS function included analysis of the intergenic region between the transcriptional start sites of ICP4 and ICP22/47 (49). These studies demonstrated that plasmids containing this region could replicate in transfected cells when infected with HSV-1. Systematic deletion of sequences in this region led to our present understanding of the limits of oriS. Although not demonstrated in these early studies, Stow et al. postulated that elements in the transcriptional regulatory regions flanking oriS might have an effect on oriS function (49). In this report, we present evidence that these elements have a significant stimulatory effect on oriS function in transient DNA replication assays.

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Viruses and cells. The KOS strain of HSV-1 was used as the wild-type virus, and all viral DNA fragments were derived from KOS DNA. Virus was grown and assayed in Vero cells (13), which were propagated as described previously (13). Plasmids. Plasmid pOS-822, containing the intergenic region between the transcriptional start sites of the immediateearly genes ICP4 and ICP22/47 (Fig. 2A and B), was constructed by digesting pIE3CAT (13) with BamHI and cloning the resulting 822-bp fragment into pGEM7Zf(+). Plasmid pOS-230 contains the 227-bp SmaI fragment of pOS-822 inserted into pGEM7Zf(+) (Fig. 1B and 2B). Plasmid pOS-80 was constructed by digesting the 227-bp SmaI fragment with HpaII and NarI and inserting the resulting 80-bp fragment containing the minimum recognized oriS sequence into pGEM7Zf(+) (Fig. 1B, 2B, and 3A). The reference plasmid pBR325-ori-230 contains the 227-bp SmaI fragment inserted into pBR325. Plasmids in which sequences were deleted starting from the ICP4 transcriptional start site of pOS-822 (Fig. 4) were generated by digestion with HindIIl at the single HindIII site located in the polylinker flanking the intergenic region. The linearized plasmids were then incubated with Bal-31 nuclease for various lengths of time. Bal-31 nuclease-treated DNAs were blunt ended with mung bean nuclease, and Hindlll linkers were added to recircularize the plasmids. Fragments containing the desired deletions were cloned into M13mpl9 and sequenced by the dideoxynucleotide method to determine the deletion endpoints. Plasmids containing internal deletions in the intergenic region (Fig. 5A) were constructed by cleavage with BssHII (Fig. 1B), digestion with Bal-31, and incubation with mung bean nuclease to generate blunt ends. BssHII linkers were added to the deletion endpoints, and the limits of the deletion were determined by DNA sequencing in M13mpl9 as described above. The transcriptional regulatory element between the ICP4 start site and core origin was inverted as follows. The HindIIl site of deletion clone pOS-H302 (Fig. 4) was replaced with a unique SpeI site to generate plasmid pOS-S302 (Fig. 6A). Plasmid pOS-822 was digested with SacI, which cleaves the DNA at a unique site within the polylinker downstream of the transcription start site of ICP22/47. The linearized DNA was then incubated for a sufficient time with Bal-31 nuclease to delete both the ICP22/47 transcription start site and oriS. The nuclease-treated DNA was then treated with mung bean nuclease, and SpeI linkers were added. The resulting plasmid, containing only the transcriptional regulatory elements located between the ICP4 transcription start site and the core origin, was cloned in both

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FIG. 3. Replication of oriS-containing plasmids. (A) The 822-bp BamHI fragment containing the intergenic region between ICP4 and ICP22/47 as described in Fig. 2A is shown beneath the scale (in base pairs). The limits of the 80-, 227-, and 822-bp oriS-containing fragments that were cloned into pGEM7Zf(+) to generate plasmids pOS-80, pOS-230, and pOS-822, respectively, are shown beneath the diagram of the BamHI fragment. (B) Southern blots of replicated plasmid DNA. Total cellular DNA was isolated from Vero cells transfected with either vector DNA or oriS-containing plasmid DNA and superinfected with HSV-1 strain KOS at 8, 14, and 20 h postinfection. DNA was digested with EcoRI and DpnI, run on a 1% agarose gel, and hybridized to a 32P-labeled pUC8 probe. The arrow denotes the position of the replicating internal standard plasmid pBR325-ori-230. (C) Replication efficiencies of oriS-containing plasmids. Each lane from the autoradiograph shown in panel B was subjected to scanning densitometry, and curves for the test plasmid and internal standard plasmid at the various times postinfection were established. The values for replication efficiency were derived by calculating the ratio of the area under the scanning curve of each plasmid to that of the replicating internal standard as 1. Under these conditions, pOS-80 replicated only approximately 0.25-fold as efficiently as the interal standard in all tests.

orientations into plasmid pOS-S302 to yield plasmids pOS822(s) and pOS-822(inv) (Fig. 6A). Plasmids containing heterologous DNA inserts between the intergenic transcriptional regulatory sequence and the core origin were constructed by inserting blunt-ended DNA fragments from MspI-digested pBR322 DNA to which were attached SpeI linkers into the SpeI site of plasmid pOS822(s). All plasmids were propagated in Escherichia coli HB101, purified by the standard alkali method, and banded twice by CsCl equilibrium centrifugation. oriS replication assay. Vero cells (5 x 105) in 60-mm dishes

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FIG. 4. Replication of oriS-containing plasmids lacking various amounts of left-hand flanking sequences. (A) Diagram of deletion mutant genotypes. The intergenic region between ICP4 and ICP22/47 shown is described in the legend to Fig. 2A. The number of each plasmid describes the number of nucleotides remaining after Bal-31 nuclease treatment. (B) Southern blots of replicated Bal-31 deletion plasmid DNAs. Transient DNA replication assays of mutant plasmids and of the replicating internal standard plasmid pBR325-ori-230 (arrow) were performed as described in the legend to Fig. 3B. (C) Replication efficiencies of deletion mutant plasmids were calculated as described in the legend to Fig. 3C.

were transfected by the calcium phosphate method with a mixture containing test plasmid DNA, internal reference plasmid pBR325-ori-230 DNA, and salmon testis DNA (39). After 4 h of incubation at 37°C, cells were shocked with glycerol and incubated for an additional 12 to 16 h. Cells were then infected with HSV-1, strain KOS, at a multiplicity of infection of 10 PFU/cell. At the indicated time postinfection, monolayers were rinsed twice with phosphate-buffered saline and lysed by the addition of 0.5 ml of 0.6% sodium dodecyl sulfate-10 mM EDTA-10 mM Tris hydrochloride (pH 7.5)-0.4 mg of proteinase K per ml at 37°C for 10 to 12 h. Total cellular DNA was then isolated and quantified spectrophotometrically. Approximately 5 ,ug of DNA (38) was digested with DpnI to remove unreplicated input DNA and with EcoRI to linearize both test and reference plasmids. The digested DNAs were fractionated on a 1% agarose gel, transferred to nitrocellulose, and hybridized to 32P-labeled, nick-translated pUC8. Southern blots were exposed to preflashed X-ray film, and the autoradiographs were scanned with an LKB scanning densitometer. Replication efficiency is defined as the ratio of the area under the peak of the test plasmid to the area under the peak of the reference plasmid,

FIG. 5. Replication of pOS-822 deletion mutant plasmicts. tA) The 822-bp BamHI fragment containing the intergenic region between ICP4 and ICP22/47 is described in the legend to Fig. 2A. Beneath the diagram of the BamHI fragment are shown the limits of the deletions in plasmids derived from Bal-31 enzyme digestion at the unique BssHII site in the intergenic region of plasmid pOS-822 (Fig. 1B). The numbers in the plasmid designations indicate the number of nucleotides deleted. (B) Southern blots of replicated deletion plasmid DNAs. Transient DNA replication assays of mutant plasmids and of the replicating internal standard plasmid pBR325-ori-230 (arrow) were performed as described in the legend to Fig. 3B. (C) Replication efficiencies of deletion mutant plasmids were calculated as described in the legend to Fig. 3C.

pBR325-ori-230. Although the reference plasmid pBR325ori-230 carries the same oriS-containing fragment as pOS230, it replicates less efficiently than pOS-230 because it contains poison sequences that retard its replication in eukaryotic cells. The presence of these sequences did not affect the utility of this plasmid as a control for test plasmid replication. The slight but significant competition of the test plasmids with the reference plasmid for replication factors was taken into account in our calculations. To ensure reproducibility of the transient oriS replication assays, all assays were performed three to five times. The results of one representative assay are shown in each figure. Although the overall efficiency of plasmid replication varied from test to test, the replication efficiency of test plasmids relative to one another and to the internal control plasmid varied less than 10% from assay to assay. RESULTS DNA sequences flanking oriS stimulate replication of oriScontaining plasmids. To determine whether replication of oriS-containing plasmids was affected by the presence oriS-

STIMULATION OF oriS FUNCTION IN HSV-1

VOL. 65, 1991

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flanking sequences, we cloned HSV-1 DNA fragments containing oriS and all (pOS-822) or portions (pOS-230, pOS-80) of the intergenic region between the 5' transcriptional start sites of ICP4 and ICP22/47 into a common genetic background, pGEM7Zf(+) (Fig. 1B, 2B, and 3A). Vero cells were cotransfected with each of the oriS-containing plasmids together with a reference plasmid, pBR325-ori-230, which contains a 227-bp fragment of oriS. The reference plasmid included to control for efficiency of transfection and to internal standard in the calculation of replication efficiencies. Although the reference and test plasmids would be expected to compete for HSV-1 replication machinery, competition between the test and reference plasmids at the molar concentrations of the two plasmids used in these tests was

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The results of replication assays with the three test plasmids are shown in Fig. 3B and C. When replication efficiency was plotted as a function of time, the replication rates of all three plasmids were linear through 20 h postinfection (data

2605

not shown). Of the three plasmids, pOS-822, which contains the entire intergenic region between the transcriptional start sites of ICP4 and ICP22/47, replicated 16- to 22-fold more efficiently than the reference plasmid at all three times tested, 8, 14, and 20 h postinfection. (Note: plasmid pOS-822 migrates to approximately the same position as plasmids pOS-80 and pOS-230, due to the presence of an additional EcoRI site in the intergenic region that releases a 710-bp fragment from the vector.) Plasmid pOS-230, which contains the same 227-bp fragment as the reference plasmid, replicated less efficiently than pOS-822 but four- to sevenfold more efficiently than the reference plasmid at all times tested. The level of replication of plasmid pOS-80, which contains only the core origin, as previously defined by others (11, 31), replicated fourfold less efficiently than the reference plasmid through 20 h postinfection. From these results, we conclude that the presence of elements in the HSV-1 immediate-early transcriptional regulatory regions flanking oriS stimulate replication from oriS by at least 80-fold relative to an oriS-containing plasmid (pOS-80) lacking these elements. Because of the consistency and reproducibility of these tests, and because of the linearity of the response over time, all subsequent tests were conducted at 8 h postinfection, when the rate of viral DNA replication is high and when DNA synthesis is most synchronous in infected cells. To further assess the role of sequences to the left of the core origin in mediating the stimulatory effect on oriS, systematic deletions were made in this region starting from the ICP4 transcriptional start site (Fig. 4). Deletions were made in these sequences because the great majority of the regulatory elements controlling the expression of both ICP4 and ICP22/47 are located between the core origin and the ICP4 transcriptional start site (40, 47). Figure 4A shows the HSV DNA sequence content of the deletion mutants generated, and the results of oriS replication assays are shown for each mutant in Fig. 4B and C. Progressive deletion of sequences in the intergenic region beginning with the ICP4 transcriptional start site (pOS-H775) and the TATA element (pOS-H704) resulted in minor but reproducible reductions in replication efficiency, whereas plasmid pOS-H608, which also lacks sequences downstream of the first TAATGARAT and CCAAT box-like binding site nearest the ICP4 transcriptional start site (Fig. 2B), reproducibly replicated to nearly wild-type levels. Deletion of sequences from the ICP4 transcriptional start site through the second TAATGARAT element in plasmid pOS-H517 produced a minor but reproducible decrease in replication efficiency. Plasmids pOSH472 and pOS-H443, which lack more than half of the sequences between the core origin and the ICP4 transcriptional start site, replicated only half as efficiently as the wild-type plasmid. Notably, nearly wild-type levels of replication were reproducibly demonstrated with plasmid pOSH389, which lacks all elements except 110 bp immediately to the left of the core origin. This result was obtained in five independent tests. Plasmid pOS-H302, which retains only the core origin, right-hand flanking sequences, and the ICP22/47 start site, exhibited a fivefold reduction in replication efficiency relative to the wild-type plasmid pOS-822. The results of these tests demonstrate the stimulatory effect of origin-flanking sequences on oriS function and indicate that the more oriS-flanking sequences were included in the test plasmid, the more efficiently the plasmid replicated. This observation is contrary to the generally accepted axiom that the longer the replicating sequence, the less efficient is plasmid replication. Thus, specific elements in oriS-flanking sequences must have a stimulatory effect on

2606

WONG AND SCHAFFER

replication. The periodicity in replication efficiency observed with these mutants may correspond with the binding of individual transcription factors, differences in the efficiency of transcription from the ICP22/47 promoter, or the fact that the deletion mutations place sequences normally at a distance from oriS adjacent to the core origin. Because plasmid pOS-H389 yielded nearly wild-type levels of origin function whereas pOS-H302 yielded greatly reduced levels, we wished to examine the sequences between positions 389 and 302 in greater detail. Progressive deletion of sequences from the convenient BssHII site towards the origin are represented by mutant plasmids pOS-Bd45, pOS-Bd75, and pOS-Bdll6 (Fig. 5A). This region contains three copies of the sequence element CCCGT TGG and two copies of a closely related sequence, CCCT TGGG (Fig. 2). The former element, first described by Whitton and Clements (52), is conserved in the intergenic region between ICP4 and ICP22/47 in both HSV-1 and HSV-2. This element resembles but is not identical to the binding site recognized by CCAAT box transcription factors (5, 10, 18, 43). As shown in Fig. 5, deletion of a single CCCGTTGG element (pOS-Bd45) resulted in a slight reduction in oriS replication efficiency relative to pOS-822, whereas deletion of two elements (pOS-Bd75) decreased replication efficiency by at least one-third. Deletion of all three elements resulted in nearly 50% reduction in replication efficiency compared with the wild-type plasmid. The deletion in plasmid pOS-Bdl55 removes some sequences in the core origin, and therefore it replicated inefficiently as expected. Progressive deletion of CAAT-like elements decreased replication efficiency significantly but not to the level shown in Fig. 4 (pOS-H302 versus pOSH389). This difference can only be due to the contribution of sequences to the left of the BssHII restriction site. In the experiments shown in Fig. 5, these sequences were brought progressively closer to the core origin. The numerous recognized cis-acting elements in these sequences may well have an enhancing effect on origin function as they are brought nearer the core origin. Together, the results presented in Fig. 4 and 5 suggest that CCCGTTGG sequence elements as well as sequences to the left of the BssHII site may be involved in the stimulation of oriS replication. To determine the precise roles of these elements in oriS function, substitution mutations in these elements are currently being generated. Distance between the core origin and left-hand flanking sequences affects the efficiency of oriS function. In order to evaluate the contribution of distance between oriS and left-hand flanking sequences on the efficiency of oriS function, heterologous DNA from plasmid pBR322 was introduced into the wild-type plasmid (pOS-822) at position 302 (plasmid pOS-H302, Fig. 4A). Mutant plasmids containing inserts of 10, 110, 217, 320, 434, and 640 bp of heterologous DNA were assayed for oriS replication efficiency (Fig. 6). Although the insertion of 10 bp had only a minor effect on oriS function, insertion of 110 bp resulted in a greater reduction in oriS function. The efficiencies of origin function of plasmids containing heterologous inserts ranging from 217 to 640 bp were approximately equal, exhibiting only -30% of wild-type plasmid replication efficiency. These results show that the insertion of sequences of 217 bp or greater had equally deleterious effects on oriS function. There was no difference in the replication efficiencies of pOS-H302 (which lacks all left-hand flanking sequences) and plasmids containing insertions ranging from 217 to 640 bp, indicating that at a

J. VIROL.

distance of at least 217 bp, the stimulatory effect of the flanking sequences on oriS function is lost. The reduction in origin function observed following insertion of nonspecific DNA between the core origin and the left-hand flanking sequences demonstrates that the distance between the left-hand transcriptional regulatory region and the core origin affects oriS function significantly. In addition to illustrating the effects on oriS function of the insertion of heterologous DNA between the core origin and left-hand flanking sequences, Fig. 6 illustrates the effect of inversion of these flanking sequences on origin function. Relative to plasmid pOS-822, in which flanking sequences are in the wild-type orientation, pOS-822(inv) exhibited a 50% reduction in origin function. This result, together with those obtained with the insertion mutants, further supports a stimulatory role for oriS-flanking sequences on oriS function and demonstrate that the effect of these sequences on origin function is both distance and orientation specific. Heterologous herpesvirus immediate-early transcriptional regulatory elements can substitute for HSV-1 oriS-flanking sequences in stimulation of oriS replication efficiency. To determine whether stimulation of DNA replication from oriS is specific to the transcriptional regulatory region controlling the expression of HSV-1 ICP4 and ICP22/47, we replaced this region with the transcriptional regulatory region of the human cytomegalovirus (HCMV) immediate-early gene IEl (3). Figure 7 illustrates the effects of this substitution on oriS function. Because not all HCMV origins of DNA replication have been mapped, we first assayed the ability of pUCCMV, which contains the HCMV immediate-early transcriptional regulatory element, to replicate in our system. As shown in Fig. 7B and C, plasmid pUC-CMV did not replicate in this system, indicating that it contains no origin that is responsive to superinfection with HSV-1. By contrast, plasmid pOS-CMV4, which contains the HCMV immediateearly regulatory element in the same orientation as the HSV-1 immediate-early regulatory element in plasmid pOS822, replicated to 90% of wild-type (pOS-822) levels. Inversion of the HCMV regulatory element (pOS-CMV3) resulted in only a minor change in the efficiency of oriS function relative to pOS-CMV4. Notably, both pOS-CMV3 and pOSCMV4 exhibited significantly greater replication efficiencies than did pOS-H302, which lacks nearly all left-hand flanking sequences. Thus, the HCMV immediate-early regulatory element can substitute for the HSV-1 immediate-early regulatory element in the enhancement of oriS replication efficiency. Whether the two regulatory elements stimulate oriS function by the same or different mechanisms is not known; however, the HCMV immediate-early regulatory element, like its HSV-1 counterpart, contains numerous binding sites for recognized cellular transcription factors. If indeed transcriptional complexes play a role in stimulation of oriS, it is not surprising that the HCMV element can substitute functionally for the HSV-1 element in origin replication tests. Unlike the HSV-1 element, however, the HCMV element was capable of enhancing replication in either orientation. This suggests the possibility that sequences critical for stimulation of oriS activity are evenly distributed within the HCMV immediate-early regulatory element, whereas those in the HSV-1 element are not. Factors that bind to oriS-flanking sequences enhance origin function. The results of the studies described above indicate that the efficiency of oriS-containing plasmids is affected by transcriptional regulatory elements adjacent to the core origin. Additionally, the fact that substitution of this element by a heterologous herpesvirus regulatory element, the

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VOL. 65, 1991

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HCMV IEl regulatory element, resulted in nearly wild-type levels of oriS function demonstrates that a homologous regulatory element is not required for efficient plasmid replication. Rather, these results suggest that protein-DNA complexes associated with origin-flanking sequences likely affect origin function. To examine the role that trans-acting factors play in oriS function, we selectively deleted the core origin from plasmid pOS-822 to yield plasmid poriS(-), which lacks oriS function yet retains nearly all cis-acting regulatory elements flanking oriS (Fig. 8A). This plasmid was used to compete with plasmid pOS-822 for factors that bind to flanking sequences in transient DNA replication assays. Figures 8B and C illustrate the results of such tests when the plasmid poriS(-) was cotransfected in 25-, 50-, and 100-fold molar excess relative to the test plasmid, pOS-822. The internal control plasmid pBR325-ori-230 was not included in this experiment because cis-acting elements in this plasmid would also compete with pOS-822 for specific DNA-binding factors. To ensure sequence specificity of the HSV-1 DNA in the competitor plasmid poriS(-), vector DNA was included in each transfection.

In four independent experiments, the addition of a 25-fold molar excess of the competitor plasmid reduced replication of plasmid pOS-822 to approximately 30% of the level observed without competitor, and addition of the competitor to a 100-fold molar excess reduced replication of the test plasmid to approximately 10% of the control level. These results demonstrate that the binding of trans-acting factors to cis-acting elements within the intergenic regulatory region is essential for maximum replication of an HSV-1 oriScontaining plasmid. DISCUSSION

HSV-1 oriS: the origin function test. By analogy to SV40, adenovirus, and other DNA-containing viruses, the proximity of HSV-1 oriS to a variety of transcriptional regulatory complexes containing both viral and cellular proteins suggests the possibility that complexes affect origin function. In order to test this possibility, we measured the effects of a variety of mutations within sequences flanking oriS on oriS replication efficiency. To ensure an identical genetic context for these analyses, origin-containing HSV-1 DNA fragments

WONG AND SCHAFFER

2608

J. VIROL.

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FIG. 8. Effect of competition of oriS-flanking sequences on the replication of the oriS-containing plasmid pOS-822. (A) The 822-bp BamHI fragment containing the intergenic region between ICP4 and ICP22/47 is shown in Fig. 2A. Also shown is the wild-type HSV-1 oriS plasmid pOS-822. The oriS deletion mutant poriS(-) was constructed by partial digestion of pOS-822 DNA with SmaI, followed by religation. (B) Transient-replication assay of pOS-822 in the presence of increasing molar concentrations of competitor plasmid poriS(-). To exclude the possible effects of vector sequences in the competition, the total amount of vector DNA in each transfection was the same. Assays were performed as described in the legend to Fig. 3B, and cells were harvested at 8 h postinfection. The replicating internal standard plasmid pBR325-ori-230 was not included in these assays. (C) Replication efficiency of plasmid pOS-822 in the presence of different concentrations of competitor plasmid. Percentages were calculated by scanning densitometry and comparison of the area under the peak produced by each concentration of competitor to the control peak without the competitor.

bearing mutations adjacent to the core origin were subcloned into the same plasmid vector. To overcome inherent variations in transient DNA replication assays that might complicate accurate quantitation of mutant plasmid replication, a replicating internal control plasmid consisting of oriS sequences in a different plasmid vector was included for direct comparison in all transfections. Our data clearly indicate that the existence of cis-acting elements adjacent to oriS affects the overall efficiency of origin-dependent DNA replication. Moreover replication efficiency was shown to be affected by the protein factors which bind to these cis-acting elements. These requirements for replication from HSV-1 oriS are consistent with those of other DNA-containing animal viruses, such as SV40, polyomavirus, bovine papillomavirus, adenovirus, and EpsteinBarr virus (8, 15, 17, 23, 24, 26, 27, 32, 34, 36, 44, 48, 53).

Based on our knowledge of origin function in SV40 and other DNA-containing viruses and on the demonstration herein that HSV-1 origin-flanking sequences stimulate oriS function in transient assays, one must ask whether oriS-flanking sequences affect origin function in the context of the viral genome and, if so, how these sequences contribute to the underlying mechanism of initiation of HSV-1 DNA replication. Efforts are under way to transfer selected mutations into the HSV-1 genome. Given that oriS is both diploid and essential for virus replication, construction of the desired viral mutants is not straightforward. Preliminary evidence, however, indicates that replication-competent mutant viruses with mutations in oriS-flanking sequences can be isolated. In the absence of such mutant viruses, our studies were conducted with an approach used previously to define the limits of HSV-1 oriS. Thus, Stow et al. demonstrated by deletion analysis of the intergenic region between ICP4 and ICP22/47 that the minimal origin was contained within a 90-bp DNA fragment (49). Although these authors did not observe the stimulatory effect of flanking sequences on oriS function, they postulated that this might in fact be the case. One may well ask why we observed the stimulatory effect whereas Stow et al. (49) did not. Aside from differences in the cell types used in the two studies, three possible explanations come to mind. First, in the present study but not in the earlier study, an internal replicating plasmid was included to aid in the precise measurement of replication efficiency and to serve as a control for transfection efficiency. Second, this study used a quantitative approach to measuring origin function, whereas the study of Stow et al. used a qualitative approach to identify the minimal origin (49). Lastly, differences in assay procedures may account for this discrepancy. In the study of Stow et al., DNA replication was assessed at 18 h postinfection, whereas in this study replication was assessed at 8 h postinfection. It is possible that the replication rate of pOS-822 is reduced by 18 h postinfection and/or that plasmids that replicate less efficiently eventually attain the same level of replication as efficiently replicating plasmids at 18 h. Differences in the replication rates of SV40 origin-containing plasmids as a function of time postinfection have been observed (21). In our hands, however, the replication rates of pOS-822 and pOS-230 were linear through 20 h postinfection. The differences in the results obtained in this study and that of Stow et al. may also reflect differences in the relative times of transfection and infection. If this were the case, the discrepancy in the outcomes could be explained by nucleosome exclusion. Previous studies of SV40 have demonstrated that once in the nucleus, transfected DNA is associated with nucleosomes as it is assembled into chromatin (6, 27). However, specific regions of transfected DNA were found to be devoid of nucleosomes. These regions corresponded to the SV40 promoter/regulatory elements and origin of DNA replication. When transferred to other sites in the SV40 genome, these elements remained nucleosome-free at these sites (27). In their study, Stow et al. infected baby hamster kidney (BHK) cells 4 h following dimethyl sulfoxide (DMSO) shock, whereas in this study we infected Vero cells 16 h after glycerol shock. At 4 h post-DMSO shock, transfected plasmid DNA may still be devoid of nucleosomes, a situation in which no origin-containing plasmid would be at a competitive advantage over any other origin-containing plasmid. By contrast, at 16 h posttransfection, association of specific plasmid sequences with nucleosomes could have far-reaching effects on the replication efficiency of origin-

VOL. 65, 1991

containing plasmids. In order to evaluate differences in the time between transfection and infection as a possible explanation for the disparity in the two studies, Vero cells were transfected with plasmids pOS-822 and pOS-230. At 4 h post-glycerol shock, monolayers were infected, and 8 h later the DNA from the infected cells was harvested. This procedure was identical to that of Stow et al. except that Vero cells instead of BHK cells and glycerol instead of DMSO were used. The results of this test were consistent with the findings reported above, that flanking sequences increased the efficiency of DNA replication by -20-fold (unpublished results). At present, therefore, the reasons for the discrepancy between the findings reported here and those of Stow et al. remain unclear. How do oriS-flanking sequences affect origin function? The results presented in this report demonstrate that, like other DNA-containing viruses, the promoter-regulatory elements adjacent to the core origin act to increase the overall replication efficiency of oriS-containing plasmids. In addition to the CCCGTTGG elements immediately adjacent to the core origin, other sequences far upstream of these elements also have stimulatory effects on origin function. Consequently, a multiplicity of elements is likely involved in the enhancement process, and multiple elements may well work together to provide the wild-type level of replication. The precise roles of protein-DNA complexes adjacent to origins of replication have not been determined; however, three possible functions for these complexes have been proposed. One is that they prevent nucleosome formation and hence enhance access of DNA replication proteins to the origin. Another is that complexes involved in transcription facilitate melting of the DNA duplex at the origin. The third proposes that proteins involved with transcription interact with replication proteins. Whether the proteins involved in these complexes can rightly be considered associated with transcription or DNA replication or both will be difficult to determine. Do transcription initiation complexes enhance accessibility of DNA replication proteins to oriS? The trans-acting factors that bind to the cis-acting regulatory elements may serve specifically to make oriS more accessible to proteins of the initiation complex. As mentioned above, nucleosome-free regions on the SV40 genome occur where promoters, enhancers, and the origin of DNA replication are found. A similar mechanism may also be operative in the transient assays reported herein. The binding of trans-acting factors to the transcriptional regulatory region surrounding HSV-1 oriS may affect the distribution of nucleosomes, thereby increasing the accessibility of the origin to viral and cellular initiation factors. Although this remains a distinct possibility, other mechanisms may also be operative in oriS activation. Like the numerous cellular trans-acting factors that can bind to the transcriptional regulatory element, the TATA box-binding protein TFIID can prevent nucleosome-mediated repression of a promoter (54). If inhibition of nucleosome-mediated repression by TFIID were the sole mechanism involved in the activation of HSV-1 oriS in these studies, then inversion of the ICP4 and ICP22/47 transcriptional regulatory element, which places the ICP4 TATA box adjacent to the core origin, should have resulted in substantially higher levels of oriS activity (Fig. 6B and C) (54). As shown in Fig. 6, inversion of the transcriptional regulatory region resulted in a replication efficiency approaching that of pOS-H302, which lacks the ICP4 and ICP22/47 transcriptional regulatory region. This result suggests that inhibition of nucleosome-mediated repression may not be the sole

STIMULATION OF oriS FUNCTION IN HSV-1

2609

mechanism responsible for the stimulation of replication at HSV-1 oriS observed in this study. Does transcription exert a secondary effect on origin configuration? With regard to the putative role of flanking sequences in the initiation of DNA synthesis, one hypothesis is that they act primarily to regulate transcription of the adjacent immediate-early genes and that they exert a secondary effect on oriS function because of their proximity. The immediate-early genes specifying ICP4 and ICP22/47 are transcribed shortly after infection (13). ICP4 represses transcription of immediate-early genes and activates transcription of early and late genes (13). Thus, the ICP4 protein itself is thought to downregulate transcription from the promoters of ICP4 and ICP22/47 by binding to sequences at the transcriptional start sites of these genes (13). Although transcription from the ICP4 and ICP22/47 promoters is substantially reduced when viral DNA replication begins at oriS, this region of the genome is in a transcriptionally active conformation, since transcription from these promoters can still be detected. This low level of transcription could modify the local configuration of the DNA, facilitating melting of the duplex at oriS. The enhancement of origin function by activation of transcription from origin-flanking sequences has been shown to be required in vivo for replication of bacteriophage X DNA (19) and has been observed during initiation of replication of the E. coli chromosomal origin, oriC (2). Do proteins involved in transcription and DNA replication interact? If a relationship exists between oriS-dependent DNA replication and transcription of flanking genes, it is possible that the proteins which bind to and regulate specific promoters and enhancers interact directly with DNA replication proteins. Studies of both SV40 and adenovirus suggest that such a direct interaction may well assist initiation from the core origins of these two viruses (21, 22, 45, 53). Such interactions may assist in promoting localized strand separation, as observed for SV40 (21, 22). In HSV-1, the proximity of the CCCGTTGG sequence elements to oriS suggests that the trans-acting factor(s) that forms a complex with this element may function in a manner analogous to the GC boxes of SV40 to stimulate oriS function. Consistent with this hypothesis is the observation that a protein(s) present in mock-infected cells binds to this element (unpublished results). This hypothesis is not without support, since comparison of the replication efficiencies of plasmids pOSH389 and pOS-H302 (Fig. 4B and C) demonstrates that the presence of these elements results in a significant stimulation of oriS function. Evidence to support the involvement of the CCAAT box-like transcription factors was presented in Fig. 7. The HCMV immediate-early regulatory element, which contains numerous binding sites for NF-I, can substitute in either orientation for the ICP4 and ICP22/47 transcriptional regulatory region. The uniform distribution of NF-I-binding sites and CCAAT box sequences within the HCMV immediateearly regulatory element (20, 35) may explain why this element can stimulate oriS function in the inverted orientation but the HSV-1 immediate-early regulatory element cannot. As shown in Fig. 2A and B, the majority of the CCAAT box-like elements within the ICP4 and ICP22/47 transcriptional regulatory region are located adjacent to the core origin. By inverting the ICP4 and ICP22/47 transcriptional regulatory region, the closest CCAAT box-like element is located approximately 200 bp from the origin, and the cluster of these elements (normally adjacent to the core origin) is located at a distance of approximately 400 bp. At

2610

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these distances, the stimulatory effects of the CCCGTTGG elements may no longer be effective (Fig. 6). The potential involvement of CCAAT box transcription factor(s) in HSV-1 oriS function is intriguing. Nuclear factor I, a member of the CCAAT box transcription factor family (46), is essential for adenovirus DNA replication (45, 46). Although the precise mechanism of activation of adenovirus DNA replication is not known, the binding of NF-I to its recognition site is required (45). Furthermore, structurefunction analysis of NF-I has shown that a domain required for DNA binding distinct from the domain involved in transcription is absolutely required for adenovirus DNA replication (33). Does transcription across oriS affect oriS function? An additional mechanism to explain how oriS may be activated in the viral genome has recently been proposed. HubenthalVoss and Roizman (25) have identified transcripts which traverse oriS sequences in both HSV-1 and HSV-2. It has been postulated that these transcripts function to regulate DNA replication by inducing changes in the superhelical density of the template or by serving as primers for DNA replication. Although this hypothesis cannot be discounted as a potential mechanism for enhancing DNA replication from oriS in the context of the viral genome, it does not explain the observations reported in this article because the presumed transcriptional start sites for the oriS transcripts are not present in the plasmids used in this study. The possible association between transcription of adjacent viral genes and oriS function clearly warrants further investigation. The use of viral mutants should prove especially valuable for this purpose. For example, in one study of mutations in this region of the HSV-2 genome, Smith et al. (47) reported the construction of viable HSV-2 mutants with deletions in the intergenic region between ICP4 and ICP22/ 47. These mutants induced the synthesis of substantially reduced levels of ICP4, ICP22, and ICP47 mRNA and were growth impaired relative to the wild-type virus. Whether the growth impairment was a consequence of the reduced transcription of ICP4 and ICP22/47 or of reduced oriS function or both was not determined. The introduction of selected mutations generated in this study into the HSV-1 genome together with the simultaneous assessment of transcription of ICP4, ICP22, and ICP47 and the efficiency of oriS function may provide a means of addressing this question. ACKNOWLEDGMENTS We thank Neal DeLuca and Christine E. Dabrowski for helpful discussions and Meg Kaveny and Marybeth Pilat for manuscript preparation. This investigation was supported by Public Health Service grants R01AI28537 from the National Institute of Allergy and Infectious Diseases and R37CA20260 from the National Cancer Institute. S.W.W. has been supported by Public Health Service grant 5T32CA09031 and a postdoctoral fellowship (PF-3403) from the American Cancer Society. REFERENCES 1. apRhys, C. M. J., D. M. Ciufo, E. A. O'Neill, T. J. Kelly, and G. S. Hayward. 1989. Overlapping octamer and TAATGARAT motifs in the VF65-response elements in herpes simplex virus immediate-early promoters represent independent binding sites for cellular nuclear factor III. J. Virol. 63:2798-2812. 2. Baker, T. A., and A. Kornberg. 1988. Transcriptional activation of initiation of replication from the E. coli chromosomal origin: an RNA-DNA hybrid near oriC. Cell 55:113-123. 3. Boshart, M., F. Weber, G. Jahn, K. Dorsch-Hasler, B. Fleckenstein, and W. Schaffner. 1985. A very strong enhancer is located

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