An Sp1/Sp3 Site in the Downstream Region of Varicella-Zoster Virus(VZV) oriS Influences Origin-Dependent DNA Replication andFlanking Gene Transcription and Is Important for VZV Replication InVitro and in Human Skin

Mohamed I. Khalil,a,c* Makeda Robinson,b Marvin Sommer,b Ann Arvin,b John Hay,a and William T. Ruyechana†

Department of Microbiology and Immunology, and the Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, Buffalo, New York, USAa;Departments of Pediatrics and Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USAb; and Department of Molecular Biology,National Research Center, Dokki, Cairo, Egyptc

The distribution and orientation of origin-binding protein (OBP) sites are the main architectural contrasts between varicella-zoster virus (VZV) and herpes simplex virus (HSV) origins of DNA replication (oriS). One important difference is the absence ofa downstream OBP site in VZV, raising the possibility that an alternative cis element may replace its function. Our previouswork established that Sp1, Sp3, and YY1 bind to specific sites within the downstream region of VZV oriS; we hypothesize thatone or both of these sites may be the alternative cis element(s). Here, we show that the mutation of the Sp1/Sp3 site decreasesDNA replication and transcription from the adjacent ORF62 and ORF63 promoters following superinfection with VZV. In con-trast, in the absence of DNA replication or in transfection experiments with ORF62, only ORF63 transcription is affected. YY1site mutations had no significant effect on either process. Recombinant viruses containing these mutations were then con-structed. The Sp1/Sp3 site mutant exhibited a significant decrease in virus growth in MeWo cells and in human skin xenografts,while the YY1 site mutant virus grew as well as the wild type in MeWo cells, even showing a late increase in VZV replication inskin xenografts following infection. These results suggest that the Sp1/Sp3 site plays an important role in both VZV origin-de-pendent DNA replication and ORF62 and ORF63 transcription and that, in contrast to HSV, these events are linked during virusreplication.

Varicella-zoster virus (VZV) is a ubiquitous human alphaher-pesvirus and is the causative agent of two diseases, varicella

(chickenpox) and herpes zoster (shingles). The VZV genome (125kb) contains at least 71 genes and contains two origins of DNAreplication (oriS) flanked by the ORF62 and ORF63 genes (7).VZV oriS contains a 46-bp AT-rich palindrome and three consen-sus binding sites for the VZV origin-binding protein (OBP)(ORF51). All three OBP-binding sites (boxes A, B, and C) for theVZV OBP [5=-C(G/A)TTCGCACT-3=] are upstream of the palin-drome and in the same orientation (Fig. 1). This is in contrast tothe structure of herpes simplex virus (HSV) oriS, where the OBP-binding sites (boxes I, II, and III) are located both upstream anddownstream of the AT-rich element. These binding sites are alsooriented in different directions and use different DNA strands(42).

Stow et al. (43) showed previously that the A site is absolutelyrequired for DNA replication and that the deletion of the C siteresults in a decreased level of replication in VZV. In contrast, thedeletion of the B site showed no effect on the extent of replication.Thus, the minimal VZV origin consists of the A site and the AT-rich palindrome. In contrast, all three OBP-binding sites in HSVare required for efficient DNA replication (1, 9, 13, 23, 31, 41, 46).These data suggest that the OPB binding pattern, the unwindingof the origin, and the development of the replication fork may bedifferent in VZV and HSV.

One possibility that might resolve this apparent difference isthat there is an alternative binding sequence for the VZV OBP inthis region, resulting in an overall mechanism similar to that ob-served for HSV. There is a partial OBP-binding site in the down-

stream region adjacent to a GA-rich region unique to the VZVorigin (Fig. 1). However, previous gel shift assays with recombi-nant OBP did not demonstrate an interaction (5), and this site wasnot protected in DNase I protection assays with recombinant OBP(43). In our previous work, we designated this sequence box D andshowed that the mutation of this site resulted in increased DNAreplication levels in both the presence and the absence of flankinggene expression (17). These findings essentially exclude the possi-bility of an alternative OBP site downstream from VZV oriS thatplays the role of box II in HSV oriS.

An alternative possibility is the presence of a cellular proteinthat binds downstream of the AT-rich stretch in VZV oriS andcompensates for the absence of the downstream OBP-bindingsite. Indeed, it has been shown for other herpesviruses that cellulartranscription factors can influence DNA replication. Both Ep-stein-Barr virus (EBV) and HSV require additional cellular and/orviral proteins that specifically activate origin-dependent replica-tion. In the case of EBV, the viral protein BZLF1, a transcription

Received 18 June 2012 Accepted 20 August 2012

Published ahead of print 29 August 2012

Address correspondence to Mohamed I. Khalil, mkhalil2@stanford.edu.

* Present address: Mohamed I. Khalil, Stanford University School of Medicine,Stanford, California, USA.

† Deceased.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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factor, is involved in DNA replication, and two cellular transcrip-tion factors, Sp1 and ZBP-89, interact with the viral DNA poly-merase and its processivity factor to stimulate replication (2). Inthe case of HSV, in addition to the requirement for the seven viralDNA replication factors, the cellular Sp1 and Sp3 proteins areinvolved in the DNA replication process (29). Mutations of theSp1/Sp3-binding site in HSV oriS decreased the DNA replicationefficiency by approximately 60%.

In previous work (16), we identified the binding of the cellulartranscription factors Sp1, Sp3, and YY1 to portions of the down-stream region of VZV oriS. YY1 appeared to have no effect onorigin-dependent DNA replication. However, the mutation of theSp1/Sp3 site (formerly GC box 1) ablated the binding of thesefactors, resulting in an increase in the level of replication in theabsence of the adjacent transcribable genes. This indicates thatSp1 and/or Sp3 acts to suppress VZV oriS-dependent DNA repli-cation in the absence of flanking gene expression.

In the work presented here, we investigate in detail the roleplayed by these cellular factor-binding sites in VZV origin-depen-dent DNA replication and in the expression of the oriS-flankinggenes ORF62 and ORF63. The results of VZV superinfection dem-onstrated that the Sp1/Sp3 site mutation caused a decrease in thelevel of DNA replication in the presence of flanking gene expres-sion and decreases in ORF62 and ORF63 gene transcription levelsin the absence of the DNA replication inhibitor phosphonoaceticacid (PAA). In contrast, the YY1 site mutation had no statisticallysignificant effects on DNA replication or on flanking gene expres-sion. However, in experiments done in the presence of PAA orwith ORF62 transfection, the Sp1/Sp3 site mutation inhibitedonly the expression of the ORF63 gene. A recombinant virus withthe Sp1/Sp3 mutation showed a significant decrease in virus

growth in MeWo cells, while a YY1 mutant virus grew as well asthe wild-type virus. Also, the Sp1/Sp3 site mutation decreasedvirus growth in human skin xenografts in the SCID mouse modelsignificantly at day 10 postinfection. These results suggest that theSp1/Sp3 site plays important roles in both VZV origin-dependentDNA replication and flanking gene transcription as well as inpathogenesis in human skin.

MATERIALS AND METHODSCells and viruses. MeWo cells, a human melanoma cell line that supportsthe replication of VZV, were grown in Eagle’s minimal essential mediumsupplemented with 10% fetal bovine serum, as previously described (40).VZV strain MSP (VZV-MSP) (11) and VZV strain pOka (10) were prop-agated in MeWo cell monolayers, as described previously by Lynch et al.(22) and Peng et al. (32).

Preparation of whole-cell lysates and immunoblot analysis. Whole-cell lysates of VZV-infected MeWo cells were prepared as previously de-scribed (32). Cells were grown to confluence in 100-mm petri dishes andinfected with wild-type and mutant pOka VZVs at a ratio of infected touninfected cells of 0.4:1. The infected cells were washed with phosphate-buffered saline (PBS) and then suspended in lysis buffer (50 mM Tris-HCl[pH 7.5], 0.15 M NaCl, 1 mM EDTA, 0.1% Triton X-100, and a proteaseinhibitor cocktail [Roche, Mannheim, Germany], added according to themanufacturer’s instructions). The lysates were collected and centrifugedfor 5 min, and supernatants were stored at �70°C for subsequent use.

Whole-cell lysates of VZV-infected MeWo cells were analyzed by 10%SDS-PAGE and immunoblotted for the presence of IE62 and IE63, asdescribed previously by Yang et al. (47). Antisera against full-length IE62(40) and antisera against full-length IE63 (48) were used as describedpreviously. Antibody against �-tubulin was obtained from Santa CruzBiotechnology (Santa Cruz, CA). Reactive bands were visualized by usinggoat anti-rabbit immunoglobulin G conjugated with horseradish peroxi-dase (Chemicon, Temecula, CA) in conjunction with Supersignal West

FIG 1 Description of the architectures of VZV and HSV-1 oriS origins of DNA replication and structure of the wild-type pLitmus R62/63F plasmid used in DpnIreplication and reporter gene assays. The positions of the OBP-binding-site boxes and their orientations are indicated. The vertical numbers under the VZV oriSstructure represent the nucleotide positions of the beginning and end of the oriS structure of the Dumas strain IRs copy of the origin of replication; these locationsare duplicated in TRs.

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Pico chemiluminescence substrate (Pierce, Rockford, IL). The quantifica-tion of the relative amounts of IE62 and IE63 normalized to the amountsof �-tubulin in loading controls was performed by using a Bio-Rad GS700imaging densitometer (Bio-Rad, Hercules, CA). Statistical significancewas determined by a one-way analysis of variance (ANOVA) followed byTukey’s post hoc test.

Plasmids. Plasmid pLitmus R62/63F and the parental plasmid pLitmusRen/FF were constructed as described previously by Jones et al. (15).pLitmus R62/63F contains the complete 1.5-kb intergenic region of VZVDNA between the ORF62 and ORF63 genes of strain pOka, including theVZV oriS structure, inserted between genes encoding Renilla and fireflyluciferases (Promega), respectively, so that the luciferase genes acted asreporters of ORF62 and ORF63 transcription. Plasmids containing Sp1/Sp3, YY1, and box A site-specific mutations within the oriS region weregenerated by mutating wild-type pLitmus R62/63F plasmids using theQuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) andthe following primer sets: 5=-ATGTCGCGGTTTTATGGGGTGTTCGCGGGCTTTTCACAGAATATA-3= and 5=-TATATTCTGTGAAAAGCCCGCGAACACCCCATAAAACCGCGACAT-3= for the Sp1/Sp3 site muta-tion, 5=-ACAGAATATATATATTCCAAATTTAGCGGCAGGCTTTTTAAAATC-3= and 5=-GATTTTAAAAAGCCTGCCGCTAAATTTGGAATATATATATTCTGT-3= for the YY1 site mutation, and 5=-GGCATGTGTCCAACCACCGTTAAAACTTTCTTTCTATATATATAT-3= and 5=-ATATATATATAGAAAGAAAGTTTTAACGGTGGTTGGACACATGCC-3= for thebox A mutation (the underlined nucleotides are the mutated nucleotidesin each primer). All primers were synthesized by IDT (Coralville, IA). Thepositions and sequences of the mutations in the VZV oriS sequence con-tained within the pLitmus R62/63F plasmids used for transfections wereverified by sequencing at the Roswell Park Cancer Institute sequencingfacility in Buffalo, NY.

The cloning of the pCMV62 plasmid expressing wild-type ORF62 un-der the control of the cytomegalovirus (CMV) immediate-early (IE) pro-moter was described previously (33–35). The ORF62 gene was derivedfrom the EcoRI E fragment of the genome of the low-passage-numberNorth American clinical isolate Scott (44).

DpnI replication assays. MeWo cells were transfected with Lipo-fectamine reagent (Invitrogen, Carlsbad, CA) according to the manufac-turer’s instructions. Four microliters of Lipofectamine reagent was usedper microgram of transfected DNA in each transfection. In transfections,which were performed with 100-mm-diameter petri dishes, 2.1 � 106

MeWo cells per dish were seeded into 12 ml of complete growth medium.The cells were 80% confluent at the time of transfection. Three hoursbefore transfection, the medium was replaced with fresh medium. Origin-dependent DNA replication experiments were performed as describedpreviously by Stow and McMonagle (41), Stow and Davison (42), andKhalil et al. (16). The cells were transfected with 5 �g of wild-type ormutant pLitmus R62/63F plasmids. At 6 h posttransfection, cells weresuperinfected with VZV strain MSP (11) by the addition of a ratio of 0.4infected cells per 1 uninfected cell to each monolayer. Total cellular DNAwas prepared at 48 h after superinfection, and the DNA was isolated byphenol-chloroform extraction followed by ethanol precipitation. TheDNA was digested with DpnI and EcoRI, as described previously by Stowand Davison (42), and analyzed by Southern blot hybridization. Transferswere done by using TurboBlotter kits obtained from Whatman, Inc. (San-ford, ME). The blots were probed with a 476-bp PCR product preparedfrom pLitmus R62/63F by using primers 5=-TAGGCCACCACTTCAAGAACTCTGT-3= and 5=-AGCAAAAGGCCAGCAAAAGGCCAGG-3=,and the probe was end labeled with[�-32P]ATP by using T4 kinase (Invit-rogen, Carlsbad, CA).

The resulting bands were quantified by PhosphorImager (MolecularDynamics, Sunnyvale, CA) analysis. The ratio of replicated plasmid toinput plasmid represents the replication efficiency of the test plasmid. Thedata from representative experiments are presented as the means of datafrom triplicate DpnI replication assays. Statistical significance was deter-mined by a one-way ANOVA followed by Tukey’s post hoc test.

Total virus DNA level determination. MeWo cells were grown toconfluence in 100-mm petri dishes and infected with wild-type and mu-tant pOka VZVs at a ratio of infected to uninfected cells of 0.4:1. Totalcellular DNA was prepared at 24 h after superinfection, and the DNA wasisolated by phenol-chloroform extraction followed by ethanol precipita-tion. For total virus DNA level determinations, the DNA was digested withHindIII and EcoRI and analyzed by Southern blot hybridization. Theblots were probed with a 337-bp PCR product containing the intergenicregion between VZV ORF3 and ORF4 and prepared by using the follow-ing primers: 5=-ATTAAACGTTCGGTACACGTCTGGT-3= and 5=-AAATAAAAAATACCTTTTTCATGCT-3=. For the control experiment to as-sess total applied cellular DNA, the DNA was digested with XbaI andEcoRI and analyzed by Southern blot hybridization. The blots wereprobed with a 744-bp PCR product containing a portion of the codingsequence of the E2F3 gene and prepared by using the following primers:5=-CAATAAATACGGCATTACATTATGA-3= and 5=-GCAGCGGCCATCTCCCACTGGGAAT-3=. Both probes were end labeled with [�-32P]ATPby using T4 kinase (Invitrogen, Carlsbad, CA). Transfers were done byusing TurboBlotter kits obtained from Whatman, Inc. (Sanford, ME).

The resulting bands were quantified by PhosphorImager (MolecularDynamics, Sunnyvale, CA) analysis. The total virus DNA level was nor-malized to the total applied cellular DNA level. Data from representativeexperiments are presented as the means of data from triplicate experi-ments. Statistical significance was determined by a one-way ANOVA fol-lowed by Tukey’s post hoc test.

Reporter gene assays. Luciferase reporter gene assays were performedas previously described (47), using MeWo cells. Transfections were per-formed by using 12-well plates. Briefly, 2 � 105 cells were seeded into eachwell 24 h before transfection. The pCMV · SPORT · �-Gal (�-galactosi-dase) vector (Gibco, Carlsbad, CA) was used as an internal control re-porter for transfections. One microgram of each reporter vector (pLitmusR62/63F) and 0.4 �g of a �-galactosidase-expressing plasmid were trans-fected for each assay with Lipofectamine reagent (Invitrogen, Carlsbad,CA), according to the manufacturer’s instructions, to act as a control forthe transfection efficiency. The cells were superinfected at 24 h after trans-fection with VZV strain MSP (11) by the addition of a ratio of 0.4 infectedcells per 1 uninfected cell to each monolayer when experiments with VZVsuperinfection were required.

In the experiments done in the context of ORF62 transfection, thereporter plasmids were cotransfected along with 0.005 to 0.02 �g ofpCMV-ORF62 plasmids. Dual-luciferase activities were normalized to thebeta-galactosidase activities. Various amounts of the pcDNA empty clon-ing vector were transfected along with plasmid pCMV62 to equalize theamounts of both total DNA and the CMV promoter in each set of trans-fections.

The cells were collected at 48 h after superinfection or transfection andlysed in 250 �l of lysis buffer (50 mM HEPES [pH 7.4], 250 mM NaCl, 1%NP-40, 1 mM EDTA). Control experiments without infection were donefor each plasmid to determine the basal expression levels. Dual-luciferaseassays were performed by using a dual-luciferase reporter assay system(Promega) with 20 �l of cell extract, 50 �l of LARII reagent, and 50 �l ofStop & Glo reagent in each assay mixture. �-Gal assays were performed byusing a �-Gal assay kit (Invitrogen) according to the microtiter plateprotocol recommended by the manufacturer. Transfection experimentswere repeated at least three times, and each set of transfection conditionsin a given experiment was performed in triplicate. The data from repre-sentative experiments are presented as the means of data from triplicategene reporter assays. Error bars indicate standard errors.

Generation of pOka recombinant viruses with Sp1/Sp3 and YY1 sitemutations. Recombinant viruses were generated by using cosmids de-rived from pOka (30). The entire pOka genome is covered in four over-lapping cosmids, designated Fsp73 (pOka nucleotides [nt] 1 to 33128),Spe14 (pOka nt 21795 to 61868), Pme2 (pOka nt 53755 to 96035), andSpe23 (pOka nt 94055 to 125124). The oriS is located in the internal repeatsequence/terminal repeat sequence (IRs/TRs) regions in cosmid Spe23.

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Mutations of the Sp1/Sp3 and YY1 sites were first introduced into pLit-mus(ORF59-65) and pLitmus(ORF66-71) and were sequenced to verifythe mutations. To insert the promoter mutations into the pSpe23 cosmid,an NheI/AvrII fragment was excised from plasmid pLitmus(ORF59-65)and ligated into pSpe23. In the final step, to insert the mutation into theother copy of the promoter, an AscI/AvrII fragment from plasmid pLit-mus(ORF66-71) was ligated into the corresponding mutant pSpe23 cos-mid.

Recombinant viruses were isolated by the transfection of melanomacells with either the wild-type or the mutated Spe23 cosmid and the otherthree intact cosmids, Fsp73, Spe14, and Pme2. To confirm the targetedmutations in the Sp1/Sp3 and YY1 sites, genomic DNA was extractedfrom virus-infected melanoma cells or HELF with DNAzol reagent (In-vitrogen, Carlsbad, CA). A PCR fragment covering the mutated regionwas amplified from genomic DNA by using Pfu polymerase (Stratagene,La Jolla, CA), gel purified with a QIAquick gel extraction kit (Qiagen, Inc.,Valencia, CA), and sequenced (Elim Biopharm, Inc., Hayward, CA).

Growth kinetics of pOka recombinant viruses with Sp1/Sp3 andYY1 site mutations. The replication kinetics of recombinant viruses wereassessed by an infectious-focus assay with immunostaining to detectplaques, as previously described (4, 24).

Briefly, 6-well assay plates and 24-well titer plates were seeded with

MeWo cells. Assay plates were incubated for various times, and severaldilutions of the samples were taken for infectious-focus assays. Titer plateswere incubated for 4 days and then fixed with 4% paraformaldehyde forimmunohistochemical staining using anti-VZV human serum. Statisticaldifferences in growth kinetics were determined by Student’s t test.

Infection of human skin xenografts in SCIDhu mice. Skin xenograftswere made in homozygous CB-17scid/scid mice, using human fetal tissuesupplied by Advanced Bioscience Resources (Alameda, CA) according tofederal and state regulations (24, 25). Animal use was in accordance withthe Animal Welfare Act and was approved by the Stanford UniversityAdministrative Panel on Laboratory Animal Care. Wild-type pOka andpOka Sp1/Sp3 and YY1 mutant viruses were passed three times in primaryHELF before the inoculation of the xenografts. The infectious-virus titerwas determined for each inoculum at the time of inoculation. Skin xeno-grafts were harvested at 10 and 21 days postinoculation, and virus was

FIG 2 Results of DpnI replication assays performed using the pLitmus R62/63F plasmid and VZV-MSP superinfection. (A) Sequences of wild-type andmutant Sp1/Sp3 and YY1 sites in the downstream region of VZV oriS. Mutatednucleotides are in italic type. (B) Typical Southern blot analysis of the effectsof site-specific mutations of Sp1/Sp3 and YY1 sites. Wild type indicates thepLitmus R62/63F plasmid containing the full intergenic region between thecoding sequences of ORF62 and ORF63, including the full oriS. Sp1/Sp3 mu-tation indicates pLitmus R62/63F containing a point mutation in the Sp1/Sp3site. YY1 mutation indicates pLitmus R62/63F containing a point mutation inthe YY1 site. Box A mutation indicates pLitmus R62/63F containing a triplepoint mutation of the core CGC triplet within box A. The upper band (R)indicates the position of DpnI-resistant DNA resulting from replication inMeWo cells. The lower band (U) indicates the position of the unreplicatedinput plasmid. (C) Histogram summarizing the data from three independentDpnI replication assays analyzed at 48 h after superinfection by VZV-MSP.Error bars indicate standard errors. Statistical significance was determined bya one-way ANOVA followed by Tukey’s post hoc test.

FIG 3 Effects of DNA replication and Sp1/Sp3 and YY1 site mutations onflanking gene expression in the context of VZV-MSP superinfection. (A) Re-sults of triplicate assays comparing the effects of the presence of the Sp1/Sp3and YY1 site mutations on the expression levels of the Renilla luciferase re-porter gene present at the position of the ORF62 gene. (B) Results of triplicateassays comparing the effects of the presence of the Sp1/Sp3 and YY1 site mu-tations on the expression levels of the firefly luciferase reporter gene present atthe position of the ORF63 gene. The promoter activities resulting from thepresence of VZV-MSP superinfection are reported as the induction (n-fold) ofluciferase activity over the basal level (without infection). �PAA indicatesexperiments done in the presence of 400 �g/ml PAA. Statistical significancewas determined by a one-way ANOVA followed by Tukey’s post hoc test. Errorbars indicate standard errors.

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titrated on a MeWo cell monolayer and analyzed by an infectious-focusassay and immunohistochemistry with polyclonal anti-VZV human im-mune serum, as described above and as reported previously by Moffat etal. (24). Virus recovered from the tissues was tested by PCR and sequenc-ing to confirm the expected mutations.

RESULTSThe Sp1/Sp3 site influences origin-dependent DNA replication.In our previous work (15), we established the binding of the cel-lular transcription factors Sp1, Sp3, and YY1 to portions of thedownstream region of VZV oriS. The binding of Sp1 and Sp3 tothe Sp1/Sp3 site (formerly GC box 1) led to the suppression ofVZV origin-dependent DNA replication in DpnI replication as-says, while the presence or absence of YY1 had no significant ef-fect, based on experiments with Sp1/Sp3 and YY1 site mutations.In these experiments, we used pVO2 plasmids, which lack thepromoter elements of the flanking ORF62 and ORF63 genes (8,26, 27). The protocol involved the transfection of the pVO2plasmid containing the VZV oriS sequence into MeWo cells, fol-lowed by VZV-MSP (11) superinfection, as described previously(16, 17).

In this paper, we extend our previous study and examine theinfluence of the presence of Sp1/Sp3 and YY1 sites on origin-dependent DNA replication in the presence of flanking gene tran-scription, looking for links between these two viral functions. Forthese experiments, in place of pVO2, we employed pLitmus R62/63F plasmids, which contain the entire region between the ORF62and ORF63 genes, including VZV oriS and the promoters for thetwo genes (15) (Fig. 1). The ORF62 and ORF63 genes themselvesare replaced by two reporters, the Renilla (R) and firefly (F) lucif-erase genes, respectively. Analogous experiments were performedby the transfection of the pLitmus R62/63F plasmid into MeWocells, followed by VZV strain MSP superinfection. As presented inFig. 2B, the CGC mutation to AAA in box A showed no replicatedDNA band, indistinguishable from the negative control, as wereported previously (17).

However, in contrast to our previously reported data using the

pVO2 plasmid, the Sp1/Sp3 site mutation (Fig. 2A) in the contextof the pLitmus plasmid showed a substantial, significant decreasein the DNA replication efficiency compared to the wild-type level.On the other hand, the YY1 site mutation (Fig. 2A) showed anincrease that was not significant (P � 0.087) (Fig. 2B and C). Thedecrease in the DNA replication efficiency seen with the Sp1/Sp3site mutation in the pLitmus plasmid implies the involvement ofthis sequence in the regulation of VZV origin-dependent DNAreplication under these experimental conditions.

The Sp1/Sp3 site is involved in expression of the ORF62 andORF63 genes. To explore the involvement of the Sp1/Sp3 site ingene expression, we carried out a series of luciferase reporter geneassays to examine the effects of Sp1/Sp3 and YY1 site mutations onthe expression of the ORF62 and ORF63 genes. We studied theeffects of these mutations in the context of VZV superinfection aswell as following the transfection of the pLitmus R62/63F plasmidin the presence and absence of 400 �g/ml phosphonoacetic acid(PAA). DpnI DNA replication assays using the wild-type plasmidconfirmed that the presence of 400 �g/ml of PAA completely in-hibited DNA replication (data not shown). The results of the lu-ciferase experiments are shown in Fig. 3A and B. The activities ofthe promoter in the presence of VZV infection are reported as theinduction (n-fold) of luciferase activities compared to the basalactivity level observed without infection.

In the absence of PAA, the Sp1/Sp3 site mutation significantlydecreased the expression levels of the ORF62 and ORF63 reportergenes (�2- and 3-fold, respectively) compared to the wild-typelevel, while in the presence of PAA, the levels of activity of theORF62 and ORF63 reporters of the wild-type plasmid were re-duced 7- and 5-fold, respectively, compared to their levels in theabsence of PAA. Also, the Sp1/Sp3 site mutation reduced the levelof activity by an additional 50% compared to the wild-type level inthe presence of PAA but only with the ORF63 reporter (notORF62). In contrast, the YY1 mutation did not significantly affectthe expression levels of the two genes in the absence of PAA andhad no effect in the presence of PAA. These data indicate that the

FIG 4 Effects of Sp1/Sp3 and YY1 site mutations on flanking gene expression in the context of ORF62 transfection. (A) Results of triplicate assays assessing theeffects of the presence of the Sp1/Sp3 and YY1 site mutations on the expression levels of the Renilla luciferase reporter gene present at the position of the ORF62gene. (B) Results of triplicate assays assessing the effects of the presence of the Sp1/Sp3 and YY1 site mutations on the expression levels of the firefly luciferasereporter gene present at the position of the ORF63 gene. (C) Absorbance at 405 nm representing the �-galactosidase activity used as a normalizer for the triplicatereporter gene assays done. The promoter activities resulting from the presence of transfected ORF62 are reported as the induction (n-fold) of luciferase activityover the basal level (without ORF62 transfection). Statistical significance was determined by a one-way ANOVA followed by Tukey’s post hoc test. Error barsindicate standard errors.

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Sp1/Sp3 site influences the expression levels of both the ORF62and ORF63 genes during viral replication and confirm our previ-ously reported finding that origin-dependent DNA replication isimportant for high levels of flanking gene transcription (17). Thestatistically significant decrease in the ORF63 expression level seenin the presence of PAA using the Sp1/Sp3 mutation also suggeststhat the involvement of the Sp1/Sp3 site in ORF63 expression isseparate from its enhancement of origin-dependent DNA replica-tion.

In the next set of experiments, to explore which elements of theVZV infection process might be responsible for the effects that wehad seen, we examined the influence of Sp1/Sp3 and YY1 sitemutations on the expression levels of the ORF62 and ORF63 genesin the context of ORF62 transfection alone. IE62, encoded byORF62, is the major VZV transactivator, which has the ability toactivate most, and perhaps all, of the VZV genes (6, 26, 33–35, 38).IE62 was shown previously to activate both the ORF62 and ORF63genes in the absence of any other viral protein (18–20, 27). Thereporter vectors were cotransfected along with an ORF62-express-ing plasmid and the pCMV �-Gal-expressing control plasmid intoMeWo cell monolayers; at 48 h posttransfection, the cells werelysed, and measurements of luciferase and �-galactosidase weretaken. The results of these assays are shown in Fig. 4A and B. Levelsof luciferase activity obtained from each pLitmus reporter plasmidin the absence of ORF62 transfection represented the basal levelsfrom this plasmid and were normalized to a value of 1. The activ-ities of the reporters in the presence of ORF62 transfection arereported as the induction (n-fold) of luciferase activities in refer-ence to the basal level of activity without ORF62 transfection.

Our first finding from these experiments was that the ORF62expression level using the wild-type pLitmus reporter in the con-text of ORF62 transfection is very low compared to the level in thecontext of VZV superinfection (Fig. 3). These data suggest, notsurprisingly, the involvement of viral factors other than ORF62 inthe expression of the ORF62 gene during normal VZV replicationand also reflect the ability of IE62 to autoregulate its promoter.Second, and similarly to the reporter gene assays done in the pres-ence of PAA, the Sp1/Sp3 site mutation resulted in a decrease inthe expression level of ORF63 only and not of ORF62. On theother hand, the YY1 site mutation had no effect on the expressionof either ORF62 or ORF63 in the context of ORF62 transfection.There was no significant change in the expression level of �-Gal(used as a normalizer) by increasing amounts of IE62 (Fig. 4C).These data imply the utilization of the cellular transcription fac-tors Sp1 and Sp3 by ORF62 for ORF63 promoter activation.

Sp1/Sp3 and YY1 site mutant viruses influence VZV replica-tion in MeWo cells and in skin xenografts. Since we had shownsignificant effects of these mutations in biochemical assays, it wasnow important to study their effects on viral replication by usingmutant viruses. Two mutants were constructed, one with the Sp1/Sp3 site mutation and one with the YY1 site mutation. pSpe23cosmids containing either the Sp1/Sp3 or YY1 mutation weretransfected with the intact pFsp73, pPme2, and pSpe14 cosmids inmelanoma cells; pSpe23 containing the wild-type sequence wastransfected along with the other cosmids as a control (recombi-nant wild-type virus). Recombinant viruses, designated the pOkaSp1/Sp3 mutant and the pOka YY1 mutant, were recovered fromthe transfections. The growth kinetics of wild-type pOka and theSp1/Sp3 and YY1 mutant viruses in MeWo cells were evaluated(Fig. 5A). The titer of the Sp1/Sp3 mutant virus was significantly

lower than that of wild-type strain pOka for the length of the study(days 1 to 5). In contrast, the titer of the YY1 mutant virus wassimilar to that of wild-type strain pOka over the period of infec-tion. These results indicate that the Sp1/Sp3 site mutation, whichinfluenced origin-dependent DNA replication as well as ORF62and ORF63 expression levels, also impaired VZV replication invitro. The YY1 mutation had no effect on the virus in this system,reflecting its behavior in biochemical assays.

To determine whether Sp1/Sp3 and YY1 site mutations af-fected VZV pathogenesis, the growths of the Sp1/Sp3 and YY1pOka mutants were next compared to that of wild-type pOka inhuman skin xenografts (Fig. 5B). The titers of all virus inoculawere similar. Wild-type pOka titers were 3.66 � 103 PFU/implantat day 10 and 3.79 � 103 PFU/implant at day 21. The titer of thepOka Sp1/Sp3 site mutant was 2.72 � 103 PFU/implant at day 10,which was statistically significantly lower than that of wild-type

FIG 5 Effects of VZV pOka Sp1/Sp3 and YY1 mutations on virus replicationin MeWo cells and in skin xenografts in the SCID mouse model. (A) Growthkinetics of pOka, the pOka Sp1/Sp3 mutant, or the pOka YY1 mutant virus inMeWo cells. Cells were inoculated with wild-type and mutant viruses at 103

PFU/ml, and infectious-virus yields were determined for 5 days after inocula-tion. (B) Skin xenografts were inoculated with pOka, the pOka Sp1/Sp3 mu-tant, or the pOka YY1 mutant. Day 0 indicates the inoculum titers of eachvirus. Bars at day 10 and day 21 represent the mean titers of infectious virus standard deviations recovered from at least three xenografts harvested at thesetime points after virus inoculation. �� represents a highly significant increase(P 0.01), and ��� represents a more highly significant increase (P 0.001).

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pOka. The titer was 3.43 � 103 PFU/implant at day 21, which isslightly lower than that of wild-type strain pOka but not statisti-cally significant. The titer of the pOka YY1 site mutant, however(3.44 � 103 PFU/implant), was not statistically significantlydifferent from the titer of wild-type pOka at day 10. At day 21,however, the titer was 4.40 � 103 PFU/implant at day 21; this isstatistically significantly higher than that of wild-type pOka. TheYY1-based difference at day 21 may reflect a growth advantageover wild-type pOka or perhaps slower growth initially, whichleaves more tissue available for infection at later times. These dataindicate that the Sp1/Sp3 site is important for VZV replication invivo and that the YY1 site may also affect growth in vivo.

Sp1/Sp3 and YY1 site mutant viruses show low IE63 expres-sion and total virus DNA levels. To assess if Sp1/Sp3 and YY1 sitemutations affected the expression levels of the ORF62 and ORF63genes flanking the oriS, the kinetics and the levels of IE62 and IE63protein expression in MeWo cells following mutant virus infec-tion were also tested. MeWo cells were infected with pOka and theSp1/Sp3 and YY1 mutant viruses and analyzed for IE62 and IE63expression levels. The expression level of IE63 with the Sp1/Sp3mutant virus was reduced significantly (about 3-fold, based ontriplicate densitometer analyses), at both 12 and 36 h postinfec-tion, compared to wild-type pOka (Fig. 6A and B). In contrast, the

level of IE62 expression did not change at these two time points(12 and 36 h) with the Sp1/Sp3 mutant virus (Fig. 6A and B). Withthe YY1 mutant virus, neither IE62 nor IE63 expression levelschanged significantly at 12 and 36 h postinfection (Fig. 6A and B).

Finally, the influence of Sp1/Sp3 and YY1 site mutations on thevirus DNA level was tested by using Southern blot analyses. Totalcellular DNA was prepared from MeWo cells infected with thewild-type and the Sp1/Sp3 and YY1 mutant pOka viruses andprobed for total virus DNA. As shown in Fig. 7A and B, the virusDNA level decreased significantly (about 2-fold) in the Sp1/Sp3site mutant virus at 24 h postinfection compared to the wild-typelevel. On the other hand, there was a significant increase in thetotal virus DNA level (by about 60%) with the YY1 site mutantvirus.

DISCUSSION

The architectures of the origins of DNA replication in the ge-nomes of the two closely related human alphaherpesviruses VZVand HSV exhibit significant differences (Fig. 1). While both vi-ruses contain AT-rich stretches, the numbers and positions of sitesor potential sites for interactions with the VZV or HSV OBPsrelative to these AT-rich stretches differ with respect to their num-bers, their relative orientations, and the requirements for their

FIG 6 Effects of Sp1/Sp3 and YY1 site mutations on IE62 and IE63 expression levels in MeWo cells during the course of VZV infection. Western blot analysesshow the expression levels of IE62, IE63, and �-tubulin, and the histograms summarize data from triplicate experiments done for each virus at 12 h (A) and 36h (B) postinfection. �-Tubulin was used as a loading control in the experiments. The blots were scanned by densitometry to obtain quantitative data (intriplicate). Statistical significance was determined by a one-way ANOVA followed by Tukey’s post hoc test.

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presence in origin-dependent replication. This is despite the factthat the VZV and HSV OBPs recognize essentially the same se-quence. Specifically, the box I-, box II-, and box III-binding sitesare all necessary for efficient origin-dependent DNA replication inHSV-1 replication (1, 9, 13, 23, 31, 41, 43).

In contrast, only box A is required for VZV origin-dependentreplication, with box B being dispensable and box C playing anauxiliary role (43). In addition, the arrangement and orientationof the three upstream OBP-binding sites in VZV preclude theformation of a hairpin, which is involved in OBP binding andorigin activation in HSV-1 (31). There also appear to be signifi-cant differences in the influence of DNA sequences within or nearthe origin on the expression levels of flanking genes. The presenceof HSV-1 origins was reported previously to have no influence onthe expression levels of flanking genes (29, 45). In the case of VZV,however, previous work by Jones et al. (15), using an identicalexperimental strategy involving recombinant viruses, showed thatthe presence of VZV origin sequences had a significant effect onflanking gene expression and that some elements within or nearthe minimal origin may act as cis elements in the expression offlanking genes.

The function or functions of the sequence(s) downstream ofthe AT-rich stretch within VZV oriS and the possibility that thesesequences can compensate for the absence of a downstream OBPsite were unknown prior to the work presented here. The VZVOBP does not bind to this sequence (5, 43), but the plasmids usedin the original identification of the minimal VZV oriS containedthis 125-bp downstream sequence, which included the Sp1/Sp3and YY1 sites. We therefore wished to determine if the Sp1/Sp3-and YY1-binding sites that we studied previously (16) were in-volved in origin-dependent DNA replication and/or in the expres-sions of the ORF62 and ORF63 genes, which flank VZV oriS.

The results of the DpnI replication assays using the pLitmusplasmids containing the intergenic region between ORF62 andORF63 indicated that the Sp1/Sp3 site acts as an enhancer of ori-gin-dependent DNA replication, while the YY1 site has no signif-icant role in the process. This differs from our previous findingswith the pVO2 plasmid lacking the promoter elements of theflanking genes (16); there could be several reasons for this. First,the oriS structure in the pVO2 plasmid is from the Dumas strain,while plasmid pLitmus is from the pOka strain. There is, however,no difference in the sequences of the Sp1/Sp3 and YY1 sites withinthe oriS downstream region between these two strains (10), ex-cluding the possibility of simple strain variation. However, in thepLitmus plasmid, there are the promoter elements of the ORF62and ORF63 genes, which are not present in the pVO2 plasmid.This suggests that the Sp1/Sp3 site (and, possibly, flanking genetranscription) may compensate for the absence of an OBP site inthe downstream region of VZV oriS.

We observed previously (17) that the VZV DNA polymeraseinhibitor PAA inhibited the expressions of ORF62 and ORF63 in anonlinear correlation with the decrease in DNA replication seen.In confirmation, in this study, we showed that the presence of PAAcaused 7- and 5-fold decreases in the expression levels of theORF62 and ORF63 reporters, respectively. If the decreases weredue solely to less available total plasmid, they would be of theorder of 25 to 30%. These results support and extend our previousfindings that origin-dependent DNA replication and flankinggene transcription appear to be coupled in VZV.

We followed these assays with the wild-type virus by usingSp1/Sp3 and YY1 mutant constructs. The mutation of the Sp1/Sp3-binding site within the oriS structure of HSV does not affectflanking gene transcription (29). In contrast, our results with thepLitmus dual-luciferase reporter plasmids showed that the muta-tion of the Sp1/Sp3 site negatively affected the transcription ofboth the ORF62 and ORF63 reporters under active DNA replica-tion (the absence of PAA). Expression from the ORF62 andORF63 reporters should be inhibited by about 10% if the solecause was a lower DNA replication efficiency leading to less avail-able total plasmid. The data showed that the decrease in the ex-pression level was �2- to 3-fold, suggesting that transcriptionregulated by the Sp1/Sp3 site is coupled to viral DNA replication;this regulation involves both sides of oriS in the presence of activeDNA replication. The data again confirm that active DNA repli-cation is required for efficient ORF62 and ORF63 expressions.

Interestingly, in the absence of viral DNA replication (in thepresence of PAA or in the context of ORF62 transfection alone),there was a statistically significant effect of the Sp1/Sp3 mutationon ORF63 transcription only and not on ORF62 transcription.This fits our data reported previously for the downstream box Dsite, where we showed effects on both origin-dependent DNA rep-

FIG 7 Effects of Sp1/Sp3 and YY1 site mutations on the total virus DNA levelin MeWo cells 24 h after VZV infection. (A) Southern blot analyses showingthe total virus DNA levels for wild-type, Sp1/Sp3 mutant, and YY1 site mutantvirus infections of MeWo cells. (B) Histograms summarizing data from trip-licate analyses done for each virus. An ORF3 promoter probe was used todetermine the total virus DNA level. An E2F3 probe was used in the loadingcontrol experiments. Error bars indicate standard errors. Statistical signifi-cance was determined by a one-way ANOVA followed by Tukey’s post hoc test.

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lication and flanking gene transcription (17). Furthermore, it ap-pears that the influence of the Sp1/Sp3 mutation on ORF62 ex-pression is due solely to the involvement of the Sp1/Sp3 site inorigin-dependent DNA replication and that origin-dependentDNA replication is required for efficient ORF62 expression.

Sp1/Sp3 sites have been identified in the replication origins ofother herpesviruses (e.g., EBV and HSV-1) (2, 29). Sp1, and pos-sibly other Sp family members, affects origin-dependent EBVDNA replication at the cis-acting element oriLyt (2, 12). This ele-ment consists of two essential domains, designated the upstreamand downstream components. The upstream component con-tains DNA-binding motifs for the EBV transcriptional activatorBZLF1. The downstream component is known to be the bindingsite of the cellular transcription factors ZBP-89 and Sp1, whichstimulate DNA replication. This stimulation is believed to resultfrom the recruitment of viral DNA replication proteins, since thedirect interaction of Sp1 and ZBP-89 with the viral DNA polymer-ase and its processivity factor was demonstrated previously in pro-tein binding assays (2). The HSV-1 genome contains two oriScore-adjacent regulatory (Oscar) elements, OscarL and OscarR, atthe base of the oriS palindrome. The mutation of either elementreduced oriS-dependent DNA replication. OscarL contains a con-sensus binding site for Sp1, and electrophoretic mobility shift as-says (EMSAs) and supershift experiments showed the binding ofSp1 and Sp3 to OscarL (29).

Sp1 is involved in lytic VZV replication, likely through its rolein the expressions of several important VZV promoters, includingthose for the viral glycoproteins gI and gE, the VZV major single-strand-binding protein, and the VZV DNA polymerase catalyticsubunit (3, 14, 32, 36, 37). Sp1 was shown previously to interactwith the VZV major transcription activator IE62 (28, 32), andthere is a high incidence of predicted Sp1-binding sites withinVZV promoters (37). The role of other Sp family members in VZVinfection and replication is still largely unknown, but the demon-stration of Sp3 binding to the Sp1/Sp3 site in the downstreamregion of oriS provides evidence for a role for this transcriptionfactor in the VZV life cycle (16).

In contrast to our results with the Sp1/Sp3 site, the mutation ofthe YY1 site, which ablated the binding of the YY1 protein to thedownstream region of VZV oriS, had no significant effect on ei-ther origin-dependent DNA replication or flanking gene tran-scription. YY1 is a 414-amino-acid zinc finger protein that is ca-pable of the repression and activation of gene transcription inseveral virus systems, including adeno-associated virus (AAV),human papillomavirus (HPV), parvovirus B19, HSV-1, and mu-rine leukemia virus (MuLV) (21, 39). YY1 was also shown previ-ously to inhibit HPV ori-dependent DNA replication in a cell-freereplication system (21).

The role or roles played by YY1 in VZV infection are unknown;thus, the identification of a bona fide YY1-binding site within the

FIG 8 Model for the role played by the Sp1/Sp3 site in the downstream region of VZV oriS in origin-dependent DNA replication and flanking gene expression.(A) The formation of Sp1/Sp3 complexes influences ORF63 expression at the immediate-early stage of infection. (B) The presence of Sp1/Sp3 complexes, coupledwith ORF63 expression activation, then enhances origin-dependent DNA replication and compensates for the absence of a downstream OBP box at the earlystage of infection. (C) The presence of active origin-dependent DNA replication in turn enhances both ORF62 and ORF63 expression levels at the early and latestages of infection.

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oriS downstream region site afforded the potential to identify aVZV-specific function for this ubiquitous cellular factor. The mu-tation of the YY1 site with the concomitant loss of YY1 binding,however, did not result in a statistically significant effect on repli-cation or gene expression under our experimental conditions.However, this does not eliminate the possibility that YY1 plays animportant role in VZV infection. Alternatively, YY1 could directlyaffect origin-dependent DNA replication and flanking gene tran-scription in primary cells or in specific tissues infected by VZV inthe human host. Our data in support of these possibilities includethe significant increase in mutant virus titers in human skin xeno-grafts at day 21 postinfection and the slight but significant increasein the total virus DNA level. The increase in the virus DNA levelachieved in the presence of the YY1 mutant virus might explainthe increases in virus titers seen in skin xenografts at day 21 postin-fection. The results obtained with the Sp1/Sp3 site mutation in thein vitro assays and the in vivo studies indicate that this site is im-portant not only for DNA replication and flanking gene expres-sion but also for VZV growth and pathogenesis. Together, ourfindings suggest that the Sp1/Sp3 site mutation may be a candidatefor a targeted change in pOka that would provide a geneticallydefined mechanism (low IE63 expression and virus DNA levels)for the further attenuation of vaccine strain Oka.

The data gathered on the ability of Sp1 and Sp3 to bind to theSp1/Sp3 site in the downstream region of oriS and their influenceon oriS-dependent DNA replication and flanking gene expressionlead us to propose a model for the role of the Sp1/Sp3 site in VZVinfection (Fig. 8). At times immediately after infection, endoge-nous Sp1 and Sp3 bind to the Sp1/Sp3 site. Once a sufficientamount of IE62 is synthesized, the most active phase of viral geneexpression starts, and at this stage, Sp1 and Sp3 interact physicallyand functionally with IE62 to activate the ORF63 promoter in theabsence of active DNA replication. The data supporting this stepare the involvement of the Sp1/Sp3 site in ORF63 expression in thepresence of PAA (Fig. 3) and in the context of IE62 transfectionalone (Fig. 4). The activation of ORF63 expression through theSp1/Sp3 site during the early stage of infection, coupled with theexpressions of the seven VZV replication factors, leads to the en-hancement of origin-dependent DNA replication. The data sup-porting this step are the involvement of the Sp1/Sp3 site in ORF63expression, which is required for the enhancement of origin-de-pendent DNA replication (compare our previously reported re-sults [16] to the results shown in Fig. 2). Efficient DNA replicationthen further enhances ORF62 and ORF63 expression levels duringthe early and late stages of infection. We note that in the presenceof PAA or an Sp1/Sp3 site mutation, the expressions of ORF62 andORF63 are inhibited.

Finally, one unresolved issue that may be explained by our datais the finding reported in 1986 by Stow and Davidson (42) thatHSV can utilize VZV oriS in DNA replication but that VZV can-not use an HSV origin. We have shown that the Sp1/Sp3 site isrequired for VZV oriS-dependent DNA synthesis. Perhaps, theabsence of an equivalent Sp1/Sp3 site in the downstream region ofHSV oriS may be a reason why VZV is unable to use the HSV site.

ACKNOWLEDGMENTS

This work was supported by grants AI018449, AI053846, and AI020459from the National Institutes of Health and by grants from the John R.Oishei Foundation and the National Shingles Foundation.

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