VIROLOGY 191,346-354 (1992)

The Varicella-Zoster Virus Immediate Early Protein, IE62,

Can Positively Regulate Its Cognate Promoter

L. P. PERERA,*” J. D. MOSCA,t M. SADEGHI-ZADEH,+ W. T. RUYECHAN,+ AND J. HAY+

*Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 208 14-4799; +Department of

Microbiology, School of Medicine, State University of New York, Buffalo, New York 142 14; and tRetrovirus Research Laboratory

Henry M. Jackson Foundation, 1500, East Gude Drive, Rockville, Maryland 20852

Received May 18, 1992; accepted July 29, 1992

Varicella-Zoster virus (VZV) is a neurotropic alphaherpes virus closely related to herpes simplex virus (HSV). How- ever, unlike its close relative HSV, VZV lacks a functional (u-TIF (a-gene transinducing factor) that activates the tran- scription of immediate early genes during the initial events of the virus life cycle. Hence, in the absence of a functional a-TIF, the mechanism triggering the expression of immediate early genes in VZV at present remains unclear. Accumu- lating evidence indicates that the gene product of the putative immediate early gene 0RF62 (IE62) plays a pivotal role in activating VZV genes of all three putative kinetic classes, namely immediate early ((~1, early (fi), and late (7) classes of VZV genes. In the present study, we show that IE62 can positively autoregulate its cognate promoter using a transient transfection assay, both in lymphocytes and in neural cells. In the same system, we can also demonstrate activation of the VZV IE62 promoter by HSV ICP4. By deletion analysis and oligonucleotide-directed site-specific mutagenesis we have localized specific regions in the IE62 promoter/upstream sequences that mediate inducibility by IE62 and HSV ICP4, and provide evidence that this promoter activation by these two proteins may be through different mechanisms. These data, taken together with the recent demonstration of the presence of IE62 in the VZ virion tegument (Kinching- ton, P. R., Hoagland, 1. K., Arvin, A. M., Ruyechan, W. T., and Hay, J. 1992. J. Viral. 66, 359-366) provides a possible mechanism by which the triggering of VZV gene expression occurs in the absence of a functional Al-TIF protein. o 1992 Academic Press. Inc.

INTRODUCTION

The family Herpesviridae is subdivided into three subfamilies: alphaherpesvirinae, betaherpesvirinae, and gammaherpesvirinae. The alphaherpesvirinae in- clude three important human pathogens: herpes sim- plexvirus (HSV) type 1, HSV type 2, and Varicella-Zos- ter virus (VZV). In addition, pseudorabies virus (PRV) and equine herpes virus (EHV) type 1, which cause disease in animals, are also included in this subfamily. The alphaherpes viruses are enveloped viruses with relatively large double-stranded DNA genomes. The complete nucleotide sequences of both HSV-1 and VZV have been determined (McGeoch et al., 1988; Da- vison and Scott, 1986). The emerging evidence, by di- rect analyses and by analogy to related sequences, indicates that the alphaherpes viruses are closely re- lated genetically and that their genomes are essentially collinear with respect to gene distribution and genetic organization. The regulation of gene expression in al- phaherpes viruses is an intense area of research and much of the current understanding of the gene regula-

’ To whom reprint requests should be addressed at Medical Virol- ogy Section, Laboratory of Clinical Investigation, Bldg. 10, 1 1 N228, NIAID, NIH, Bethesda, MD 20892.

tion in these viruses has been derived from the proto- type HSV-1 system.

In HSV, during productive infection, viral gene ex- pression is temporally regulated; the viral genes are expressed in three broad classes, termed immediate early ((Y), early @), and late (y) (Honess and Roizman, 1974, 1975; Clements et al., 1977; Roizman and Sears, 1990). The first set of genes to be expressed are the five immediate early genes, CUO, a4, a22, a27, and a47, and the transcription of these (Y genes by the host cell RNA polymerase II occurs in the absence of any de novo viral protein synthesis (Alwine et a/., 1974; Con- stanzo et al., 1977). It now seems clear that ICP4 (IEl75), the product of the a4 gene, plays a central role in the temporal expression of HSV genes in the in- fectious cycle of the virus. Using HSV mutants that carry either conditional or null mutations in the cu4 gene, it has been demonstrated that ICP4 is absolutely essential for the expression of early and late genes (Dixon and Schaffer, 1980; Holland et al., 1979; Knipe et al., 1978; Preston, 1979a,b; Watson and Clements, 1980). Furthermore, a number of studies have clearly shown that ICP4 down-regulates the expression of im- mediate early genes (including its own). For example, conditional lethal ICP4 mutants show accumulation of immediate early gene products under nonpermissive

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346

REGULATION OF VARICELLA-ZOSTER VIRUS GENE EXPRESSION 347

conditions (DeLuca et a/., 1985; DeLuca and Shaffer, 1988; Preston, 1979a,b). Consistent with this, the re- pression of immediate early gene promoters by ICP4 has been reproduced in transient transfection assays (Gelman and Silverstein, 1987; Roberts et al., 1988; Preston, 1979a,b; O’Hare and Hayward, 1985, 1987).

Homologues of the HSV a4 gene have been identi- fied in other alphaherpes viruses, based on DNA se- quence homology and genetic topology, and include theIE62geneofVZV,theIEl80geneofPRVandtheIE 1 of EHV-1. VZV IE62 is a protein of 1310 amino acid residues with a predicted molecular weight of 140 kDa, although the actual size of the protein appears to be approximately 175 kDa as determined by polyacryl- amide gel electrophoresis (Felser et a/., 1988; Shiraki and Hyman, 1987). It is a DNA-binding phosphoprotein and can be found in both the cytoplasm and the nu- cleus of VZV-infected cells. Furthermore, its abundant presence in thevirion tegument has been recentlydem- onstrated (Kinchington et al., 1992). Consistent with the significant sequence homology between VZV IE62 and ICP4 of HSV, these two proteins have been shown to share many functional features. Recent work from Felser et a/. (1987) has demonstrated that HSV-1 mu- tants with lesions in ICP4 can be complemented either by transfected plasmids or by functional cell lines which express VZV IE62. In addition, Disney and Ever- ett (1990) were able to create a viable HSV-1 recombi- nant virus with ORF62 in place of the coding se- quences for ICP4, thus conclusively proving significant functional homology between these two gene prod- ucts.

As an extension of the structure-function compari- sons of VZV IE62 and ICP4 of HSV, we investigated the ability of the VZV protein to autoregulate the cognate promoter activity by using a transient assay in vitro in cell types (human lymphocytes and neural cells) in which VZV has been detected in the infected patient. In this communication, we demonstrate that, in contrast to the HSV system and to VZV IE62 in VERO cells, VZV IE62 in lymphocytes and neural cells is able to aug- ment its cognate promoter activity.

MATERIALS AND METHODS

Cell lines

A CD4-positive, continuous human T cell line A3.01 (Folks et al., 1985) was obtained from the AIDS Re- search and Reference Reagent Program, NIAID, NIH. The A3.01 cells were grown in RPMI 1640 supple- mented with lOq/o fetal calf serum (FCS) and 2 mM L-glutamine. The rat pheochromocytoma PC-l 2 cell line (Green and Tischler, 1976) possessing a neural crest origin was obtained from the American Type Cul-

N S C K N X

I I 11111 I I I+ I la-m

N” f

% F II

$5 9: T: h! 7 7 F

FIG. 1. Schematic map of promoter/upstream elements of the ORF62 gene. The initiator codon is 73 bp downstream of the tran- scription start site, +l. Restriction endonuclease sites used in the construction of ORF62 promoter/upstream deletion panel are indi- cated with their positions relative to the transcription start site. Ab- breviations for the restriction endonuclease sites: N, Mel; S, Smal; C, C/al; K, Kpnl; X, Mol. The solid line represents the vector se- quences containing the Ndel site used in creating p62CATA258 (see text for details).

ture Collection. The PC-l 2 cells were grown in RPMI 1640 supplemented with 10% horse serum, 5% FCS and 2 mM L-glutamine.

Plasmid constructs

Recombinant plasmids were constructed by stan- dard procedures (Maniatis eta/., 1989). In constructing pCMV62, the EcoRl E clone (Straus et al., 1982) was digested with Bg/ll and Seal and a 5-kb fragment con- taining the entire coding region of the ORF62 gene was isolated by agarose gel electrophoresis. This 5-kb frag- ment was then cloned into pG310 (a kind gift from E. Mocarski, Stanford University, CA) that had been di- gested with EcoRl (the EcoRI-cut ends were subse- quently blunted by mung bean nuclease treatment) and BarnHI. The plasmid pG310 contains the strong constitutive promoter cassette of the immediate early (IE) gene 1 of human cytomegalovirus (HCMV). The resultant construct, pCMV62, contains the coding re- gion of VZV IE62 under the control of the HCMV IE 1 promoter.

The p62CAT plasmid was constructed by digesting pGi26 (Inchauspe et a/., 1989) containing the VZV IE62 gene with BstXl (the BstXI-cut ends were blunted by mung bean nuclease treatment) and BarnHI. A 4.2-kb fragment containing the promoter region of ORF62 was isolated by agarose gel electrophoresis. The cod- ing region of the chloramphenicol acetyl transferase (CAT) gene was isolated from the pCAT BASIC plasmid (Promega) in a 1.7-kb BarnHI-Sphl fragment, in which the Sphl-cut end had been blunted by mung bean nu- clease treatment. This fragment was then ligated to the 4.2-kb fragment containing the promoter region of IE62 isolated from pGi26. The resultant construct, p62CAT, contains the coding region for CAT (with the first translation start codon being that for CAT) under the control of ORF62 promoter/upstream elements embedded in a segment extending from -5 to -1510 bp relative to the initiator AUG codon of ORF62 (see Fig. 1).

348 PERERA ET AL.

Deletions in the promoter/upstream elements of ORF62 were made in the p62CAT construct by select- ing convenient restriction sites (see Fig. 1). To create p62CATA1139, p62CAT was digested with Smal and C/al and subsequently treated with mung bean nu- clease to blunt the cut ends. After removal of the inter- vening segment by agarose gel electrophoresis, the blunt ends were resealed by T4 DNA ligase. The re- sultant construct contains the promoter/upstream elements of VZV ORF62 embedded in a segment ex- tending from +68 to - 1 139 bp relative to the transcrip- tion initiator site (+l) of the ORF62. To create p62CATA578, p62CAT was digested with Smal and Kpnl and subsequently treated with mung bean nu- clease to blunt the cut ends. After removal of the inter- vening segments by agarose gel electrophoresis, the blunt ends were resealed with T4 DNA ligase. The re- sultant construct contains the promoter/upstream ele- ments of VZV ORF62 embedded in a segment extend- ing from +68 to -578 bp relative to the transcription initiation site (+l) of ORF62. To create p62CATA258, the p62CAT was digested with Noel. After removal of the intervening segment by agarose gel electrophore- sis, the ends were resealed by T4 DNA ligase. The resultant construct contains the promoter/upstream el- ements of VZV ORF62 embedded in a segment ex- tending from +68 to -258 bp relative to the transcrip- tion initiation site (+l) of ORF62. To create p62CATA124, p62CAT was digested with Smal and Xhol and the cut ends subsequently blunted by treating with mung bean nuclease. After removal of the inter- vening segment by agarose gel electrophoresis, the ends were resealed by T4 DNA ligase. The resultant construct contains the promoter/upstream elements of VZV ORF62 embedded in a segment extending from +68 to -124 bp relative to the transcription initiation site (+l) of the ORF62. All the deletion constructs of p62CAT were sequenced across the resealed joints to verify the integrity of the deletions. The plasmid p62CATA45 was created by digesting p62CATA258 with Ndel followed by Ba/31 exonuclease treatment. After extracting the DNA with phenol/chloroform and precipitating with ethanol, the ends were flushed with T4 DNA polymerase in the presence of deoxynucleo- tide triphosphates (NTP) and religated with T4 DNA ligase. After transformation of the ligated DNA, a clone was identified by DNA sequencing that retained the promoter/upstream elements of the VZV ORF62 gene extending from +68 to -45 relative to the transcription initiation site (+l) of IE62 and was designated p62CATA45.

The constructs pGH1 14, containing the coding re- gion of a4 gene of HSV-1 under the control of simian cytomegalovirus (SCMV) immediate early (IE) gene

promoter, and pPOH30, carrying a chimeric construct with the CAT gene under the control of promoter/up- stream elements of the cu4 gene of HSV-1, were kindly provided by G. S. Hayward, The Johns Hopkins Univer- sity, MD.

Oligonucleotide-directed, site-specific mutagenesis

To create mutations in the ICP4-binding consensus sequence ATCGTC present in the promoter/upstream elements of the VZV IE62 gene, the p62CATA258 was digested with Noel (the Ndel-cut ends were blunted with mung bean nuclease) and BarnHI. A 1.9-kb frag- ment containing the ORF62 promoter-CAT chimeric transcriptional unit was isolated by agarose gel electro- phoresis. This fragment was then ligated to HindIll (the HindIll-cut ends were blunted with mung bean nu- clease) and BamHI-digested phagemid vector PBS- (Stratagene). When infected with R408 (Russel et a/., 1986) helper phage, the noncoding strand of the ORF62 promoter-CAT segment is rescued in this re- combinant phagemid. To create substitution muta- tions in the consensus core sequence ATCGTC, a synthetic oligonucleotide with the sequence 5’CTC- GTCCAATCACTACATAJTCTTATCATTAAGAATA3’ (substituted bases underlined) corresponding to the coding strand of ORF62 was used. To create a com- plete deletion of the consensus sequence, a synthetic oligonucleotide with the sequence 5’CGTCCAATCAC- TAClTATCAlTAAGAATATACACGG3’ correspond- ing to the coding strand of ORF62 was used. The oligo- nucleotide-directed, site-specific mutagenesis was performed using the Muta-Gene in vitro mutagenesis kit (Bio-Rad) according to published procedures (Geis- selsoder et al., 1987).

DNA sequencing

CsCl density gradient-purified plasmid DNA was se- quenced using the Sequenase DNA sequencing kit (U.S. Biochemical) according to published procedures for sequencing double-stranded DNA (Perera et a/., 1991).

DNA transfections

In transient transfection assays, 10 pg of target con- struct DNA was used in all the experiments. The effec- tor DNA was also used at 10 pg in cotransfection ex- periments, except in effector titration experiments, where up to 100 pg of effector construct DNA was used. In experiments where no effector construct DNA was used, 10 pg of pGEM3Z (Promega) plasmid DNA was added to keep the total amount of DNA constant at 20 rg per transfection. For electroporation of A3.01 cells, cells in log-phase growth were resuspended in

REGULATION OF VARICELLA-ZOSTER VIRUS GENE EXPRESSION 349

RPM1 1640 medium with 209/o FCS but without gluta- mine or antibiotics, at a density of 5 X 10’ cells per milliliter. Aliquots (350 ~1) of the cell suspension were mixed with plasmid DNA in sterile 1.5-ml cr-yovials (Nunc) and incubated on ice for 15 min. The cells were then transferred into an electroporation cuvette with a 0.4-cm electrode gap and was electroporated with a single pulse; we used a Gene-Pulser electroporator (Bio-Rad), with the settings at 0.20 volts and 960 PF with a capacitance extender. For electroporation of PC-l 2 cells, the cells in log-phase growth were resus- pended in RPMI 1640 with 20% FCS (after passing through a 20-gauge needle to obtain a clump-free cell suspension) and electroporated with a single pulse at 0.22 volts and 960 PF with a capacitance extender. After pulsing, the cells were immediately transferred into 24-well tissue culture plates that had been chilled on ice and incubated for a further 15 min on ice, before adding 1 ml of growth medium. The cells were then grown for 48 hr before harvesting.

CAT assays

Cells were harvested 48 hr after DNA transfection and CAT assays were performed essentially as de- scribed by Gorman et a/. (1982). Cells were washed once with phosphate-buffered saline (PBS) and resus- pended in 0.25 lI/I Tris-HCI (pH 7.8) and disrupted by three cycles of freeze-thawing. Protein concentrations in cell lysates were determined using the Bio-Rad pro- tein assay kit according to the manufacturer’s instruc- tions. The CAT activity was assayed by using the same amount of total protein for all samples in an individual experiment. The CAT activity was quantitated using a Phosphorlmager scanner with Image&ant software from Molecular Dynamics (Sunnyvale, CA). All experi- ments were repeated at least three times with indepen- dent DNA transfections.

RESULTS

We recently demonstrated the ability of the putative VZV immediate early protein IE62 to activate represen- tative genes of VZV that belong to all three predicted kinetic classes (Perera et a/., 1992) providing evidence to confirm this protein as a major VZV regulatory pro- tein. IE62 has recently been shown also to be a VZV structural protein and, as an approach to determining a possible role for IE62 in the initial stages of VZV infec- tion, we have, in the present study, evaluated the ef- fects of IE62 on its cognate promoter using a transient transfection assay. The expression of IE62 from the strong constitutive IE-1 promoter of HCMV (pCMV62) enabled us to assess effects on its cognate promoter, avoiding promoter competition interference.

Fold induction 1 12.8 6.2 4.2 2.6

p62CAT I I

pCMV62 ON lwl 2wl 5ovg 1wKl

FIG. 2. IE62 activation of its cognate promoter in A3.01 cells. The target construct p62CAT (10 pg) was cotransfected with different amounts of the effector construct pCMV62 by electroporation as described under Materials and Methods section. Cells were har- vested 48 hr after DNA transfection and CAT assays were per- formed. The CAT activity was quantitated using a Phosphorlmager scanner with Image&rant software from Molecular Dynamics (Sun- nyvale, CA). The fold induction of the CAT activity seen in transfec- tions with effector plasmid at varying amounts was calculated rela- tive to the control experiment in which 10 rg of pGEM3Z (Promega) DNA was used in cotransfection. The value was set equal to 1 .O in the control experiment and the fold induction relative to the control experiment is presented.

IE62 activation of its cognate promoter

As shown in Fig. 2, the IE62 protein was able to activate the cognate promoter in A3.01 cells, a perma- nent cell line of human T lymphocytes. To examine the inducibility of the IE62 promoter in a more quantitative manner, dose-response experiments were performed in which a constant amount of p62CAT (10 pg) was transfected with increasing amounts of pCMV62. The maximal activation of the cognate promoter was seen when both target (p62CAT) and the effector (pCMV62) were in equimolar concentrations although as little as 1 pg of pCMV62 was able to elicit a demonstrable in- duction of the p62CAT target (data not shown). How- ever, it should also be noted that when pCMV62 was present in molar excess, the extent of cognate pro- moter activation was diminished (Fig. 2). Furthermore, when pG310 (the vector plasmid construct containing the HCMV IE promoter cassette) was cotransfected with p62CAT, there was no demonstrable induction of the target, thus ruling out the possibility that activation seen with pCMV62 was due to titration of a cellular repressor by the HCMV IE promoter region (data not shown).

We next tested the ability of the major regulatory protein of VZV (a neurotropic virus) to activate its cog- nate promoter in the pheochromocytoma cell line, PC- 12, which has been widely used as a model system in studies of sympathetic neuronal function and develop-

350 PERERA ET AL.

Acetylatlon % 4.8 18.4 22.5 36.5 59.6

p62CAT 1 I

pCwa2 opg w9 25k9 w&l low9

FIG. 3. IE62 activation of its cognate promoter in PC-1 2 cells. The target construct p62CAT (10 rg) was cotransfected with different amounts of the effector construot pCMV62. Cells were harvested 48 hr after DNA transfection and CAT assays were performed. The CAT activity was quantitated using a Phosphorlmager scanner with Ima- ge&ant software. Values abovethe figure are the ratio of acetylated chloramphenicol to its unacetylated form, expressed as percent- ages.

ment (Green and Shooter, 1980). In PC-12 cells, as with A3.01 cells, the IE62 protein was able to activate its cognate promoter (see Fig. 3). In PC-l 2 cells, the gene product of ORF62 demonstrated a dose-depen- dent activation of the cognate promoter, although no squelching was seen with excess activator, in contrast to the A3.01 lymphocytic cell line.

HSV ICP4 activation of the IE62 promoter

Since IE62 of VZV has been shown to be functionally similar to ICP4 of HSV in many aspects, we next as- sessed the ability of the HSV protein to modulate the activity of the ORF62 promoter, using a transient co- transfection assay in the 83.01 lymphocyte cell line. As shown in Fig. 4, HSV ICP4 strongly activates the VZV ORF62 promoter. In order to determine the optimal ef- fector-target combination needed for activation, the cotransfection experiments were performed with a fixed amount of target DNA (10 pg p62CAT) and in- creasing amounts of pGH 114 (the ICP4 gene of HSV driven by the SCMV IE promoter). As with IE62, the smallest amount of pGHll4 used elicited the maximal activation of the ORF62 promoter in A3.01 cells.

IE62 activation of the HSV ICP4 promoter

Having shown that ICP4 could activate the IE62 pro- moter, we next looked at the activity of IE62 on the ICP4 promoter. As shown in Fig. 5, IE62 was able to activate the ICP4 promoter of HSV strongly, whereas the SCMV-promoter driven ICP4 construct had no de- monstrable effect on its cognate promoter. From these results it is apparent that, although IE62 may share some functional attributes with HSV ICP4, the underly-

Fold Induction 1 29.8 19.8 16.5 14.1

p62CAT 1 I

pGH114 (SCMVXP4) - WI 1wg wg 25P9

FIG. 4. HSV ICP4 activation of the IE62 promoter in A3.01 cells. The p62CAT plasmid DNA (10 pg) was cotransfected with varying amounts of the effector plasmid pGHl14 (the ICP4 gene of HSV-1 driven by the simian cytomegalovirus IE promoter). The cells were harvested 48 hr after DNA transfection and the CAT assays were performed. The fold induction of the CAT activity seen in transfec- tions with effector plasmid at varying amounts was calculated rela- tive to the control experiment in which 10 rg of pGEM32 (Promega) DNA was used in cotransfection. The value was set equal to 1 .O in the control experiment and the fold induction relative to the control experiment is presented.

ing mechanisms by which these two closely related regulatory proteins mediate gene activation may be dif- ferent.

Sequences involved in activation of the IE62 promoter

In an attempt to dissect the mechanisms by which these two proteins activate the VZV ORF62 promoter, we made a panel of deletion constructs representing varying lengths of the ORF62 promoter/upstream ele- ments linked to the CAT reporter gene. The ability of

Acetylation % 0.5 0.4 0.5 0.4 51.6 33.1 14.1

pPOH30 (a4CAT) 1 I

pGH114 (SCMWCP4) - 1wl 2wg 5opg - - -

pCMV62 - - - - w9 2w SOP9

FIG. 5. IE62 activation of the HSV ICP4 promoter in A3.01 cells. The target, pPOH30 (10 pg) carrying a chimeric construct with the CAT gene under the control of promoter elements of the (~4 (ICP4) gene of HSV-1 was transfected with varying amounts of pGH114 or pCMV62. Cells were harvested 48 hr after DNA transfection and the CAT assays were performed. The CAT activity was quantitated as described under Materials and Methods. Values above the figure are the ratio of acetylated chloramphenicol to its unacetylated form, ex- pressed as percentages.

REGULATION OF VARICELLA-ZOSTER VIRUS GENE EXPRESSION 351

Acetylatlon % 0.7 6.5 7.7 0.6 1.8 3.6 0.7 9.4 4.0

, 1 2 3 ,, 1 2 3 ,,l 2 3 ,

p62CAT p62CATA1139 p62CATA578

Acetylatlon % 0.6 9.6 10.3 2.2 a.7 6.4 0.5 0.7 10.7

, 1 2 3 ( , 1 2 3 ,, 1 2 3 ,

p62CATA258 p62CATA124 p62CATA45

FIG. 6. Sequences involved in activation of the IE62 promoter in A3.01 cells. A panel of deletion constructs of the ORF62 promoter/ upstream elements linked to the CAT reporter gene (see text for details) was cotransfected with pGEM3Z (lanes l), pGH 1 14 (lanes Z), and pCMV62 (lanes 3). In cotransfections, target and effector were used at equimolar amounts (10 pg). Cells were harvested 48 hr after DNA transfection and the CAT assays were performed. The CAT activity was quantitated as described under Materials and Methods. Values above the figure are the ratio of acetylated chlor- amphenicol to its unacetylated form, expressed as percentages.

IE62 and ICP4 to activate the deletion constructs was then examined by transient cotransfection assays in the A3.01 cells using equimolar amounts of target and effector DNA. As shown in Fig. 6, all the deletion con- structs used, including the truncated ORF62 promoter containing only 124 bp of sequence upstream from the cap site (p62CATA124) were still responsive to both IE62- and ICP4-mediated activation. However, when the truncated construct that retained only 45 bp of se- quence upstream from the cap site (p62CATA45) was used, only IE62, and not ICP4, was able to activate. Thus, this promoter activation appears to take place through mechanisms which are different for IE62 and for ICP4.

Many HSV genes whose expression is modulated by ICP4 possess in their control sequences the motif ATCGTC, that mediates high-affinity binding of ICP4 (Faber and Wilcox, 1986, 1988; Tedder et a/., 1989; Beard et a/., 1986). The IE62 promoter region contains this motif, 71 bp upstream from the TATA box (present in p62CATAl24). To test the motif’s responsiveness in the IE62 promoter, two mutant constructs were made, either by altering the ATCGTC consensus motif to ATATTC (p62CATMcu4) or by completely deleting the core consensus motif (p62CATAcz4) by oligonucleo- tide-directed, site-specific mutagenesis. As shown in

Fig. 7, when p62CATMa4 and p62CATA(u4 were tested for ICP4-mediated inducibility using equimolar amounts of target and effector DNA in a transient co- transfection assay in A3.01 cells, both mutants still re- tained the ICP4 inducibility. Thus, it appears that ICP4- mediated induction of the ORF62 promoter is not de- pendent on the high-affinity, ICP4-binding core consensus motif ATCGTC, present in the ORF62 pro- moter/upstream region.

DISCUSSION

VZV, unlike its close relative HSV, lacks a functional (r-TIF ((Y gene transinducing factor; also designated Vmw65 or VP1 6) which is a potent transactivator of (Y genes of HSV (Betterson and Roizman, 1983; Camp- bell et al., 1984; Pellett et al., 1985) and which is lo- cated in the virion tegument. It is not clear at present what triggers an analogous induction of putative VZV (Y genes (which presumably include IE62) during produc- tive infection of cells with VZV. However, Kinchington et a/., (1992) having demonstrated the presence of IE62 protein in the VZV virion tegument, proposed that this incorporation of IE62 into the virus structure may be a compensatory mechanism to ensure rapid ex- pression of cy (immediate early) genes of VZV upon entry into a susceptible cell. If this were the case, it would be consistent with the hypothesis that IE62 might positively regulate its cognate promoter in ap- propriate cell types; we already know that IE62 posi- tively regulates promoters of a number of other VZV proteins from all putative kinetic classes.

Fold Induction 1 11 30 1 12 30

, 1 2 31 I’ 2 3 ,

p62CATMwl p62CATAa4

FIG. 7. The ICP4-mediated inducibility of the ORF62 promoter does not require the ATCGTC motif. In p62CATMa4, the consensus ATCGTC motif is altered to ATAllC. In p62CATAa4, the consensus motif is completely deleted. These two mutant constructs were co- transfected with pGEM3Z (lanes 1). pGH 1 14 (lanes 2) and pCMV62 (lanes 3). In cotransfection experiments, both target and effector were used at equimolar amounts (10 pg). The fold induction of the CAT activity seen in transfections with effector plasmids was calcu- lated relative to the control experiment in which 10 fig of pGEM3Z (Promega) DNA was used in cotransfection. The value was set equal to 1 .O in the control experiment and the fold induction relative to the control experiment is presented.

352 PERERA ET AL.

A human T lymphocyte background for transient co- transfection assays was selected to evaluate the ef- fects of IE62 on its cognate promoter, since it has been well documented that during natural VZV infection, es- pecially in the prodromal and early clinical phases when the virus is actively replicating in the human host, VZV can be isolated from the peripheral blood mononu- clear cells. Among these, T lymphocytes predomi- nantly appear to be able to support VZV growth, imply- ing an important role for human T lymphocytes in VZV biology (Asano et al., 1989; Gilden et a/., 1987; Korop- chak et al., 1989, 1991; Ozaki et al., 1986; Vansover et a/., 1987). We also examined cells of neural origin, since VZV seems likely to be able to grow productively both in the central and peripheral nervous system.

Our experiments clearly show that in both T lympho- cytes and in neural cells, IE62 can activate its own promoter, consistent with the notion that IE62 may be able to play a role analogous to that of HSV a-TIF. This activation, however, depends on the amount of effec- tor plasmid added to the system, with an obvious inhibi- tory effect at high concentrations in T lymphocytes. Although the basis for this diminution in activation is not clear, it may be that superfluous accumulation of IE62 may be detrimental to functional activity. Similar squelching-type phenomena have been observed with potent transactivators in other systems (Greaves and O’Hare, 1989; Gelman and Silverstein, 1986; Gill and Ptashne, 1988). Although the profile of effector-depen- dent activation was different in the neural (PC-l 2) cells in comparison to T lymphocytes, in that no squelching was seen with excess effector, the magnitude of acti- vation seen even with the highest amount of effector (pCMV62) was only 12.4-fold compared to the basal level of expression. In the T lymphocytes, a compara- ble level of activation (12.8-fold) was seen at the lowest amount of activator used (10 pg of pCMV62). Thus, it is probable that the dose-response profiles of the cog- nate promoter activation reflect different thresholds of saturation in the two cell types. From the above results it is clear that the gene product of VZV ORF62, as well as its HSV homologue ICP4, can activate ORF62 pro- moter-directed expression.

However, in the HSV system, it is well documented that, although ICP4 transcriptionally activates both p and y genes of HSV, the cr genes, including ICP4 itself, are negatively regulated. Recently, Disney and co- workers (1990) reported that VZV IE62 similarly was able to repress its own promoter in both Vero and BHK cells. The data presented in this study show that, in T lymphocytes and in neural cells, the opposite effect can be demonstrated. Disney et a/. (1990) included the HSV (r-TIF in the cotransfection assays to boost the basal expression of the IE62 promoter to detectable

levels in their assays, and indicated that the maximal level of repression was seen under such conditions. To investigate this difference in approach, we also in- cluded HSV a-TIF in our assays in T lymphocytes. How- ever, the pattern of IE62-mediated activation of its own promoter remained unchanged, although the inclusion of a-TIF did increase the basal expression of p62CAT (data not shown).

When we looked at minimal sequence requirements for promoter activation by examining the ability of trun- cated IE62 promoter constructs to act as targets for self-activation, we found that the most truncated con- struct which can still be activated (p62CATA45) retains only 15 bp upstream of the TATA box of the ORF62 promoter. This suggests that IE62 self-activation may be mediated by a TATA sequence-dependent mecha- nism. In contrast, using the same assay, HSV ICP4-in- duced activation of the ORF62 promoter appears to require additional sequences (the 80-bp region be- tween the -124 and -45 of the ORF62 promoter), since p62CATA45 was refractory to ICP4-mediated in- ducibility. It has been demonstrated that many HSV genes modulated by ICP4 possess a consensus core motif (ATCGTC) that mediates high-affinity binding of ICP4 (Faber and Wilcox, 1986, 1988; Tedder et al., 1989; Beard eta/., 1986). However, currently not much is known about &-acting elements important in acti- vation of heterologous promoters by ICP4. Since the 80-bp region mediating the ICP4 inducibility to ORF62 promoter does contain this core motif (7 1 bp upstream of the TATA box of the ORF62 promoter) we assessed the contribution of this motif in the heterologous VZV ORF62 promoter activation. Our results showed that ATCGTC did not appear to be necessary. Recently, it has been shown that a number of other HSV genes are modulated by ICP4 in the absence of the high-affinity, ICP4-binding, core consensus motif ATCGTC (Michael et al., 1988).

Finally, when basal promoter activities of the trun- cated ORF62 promoter constructs were compared (see Fig. 6) the p62CATA124 deletion consistently demonstrated enhanced activity. This indicates that sequences further upstream than -124 may have an inhibitory effect on expression from the ORF62 pro- moter of VZV; this is currently being investigated.

In summary, we have shown that the product (IE62) of the putative immediate-early gene VZV ORF62, a major regulatory protein of VZV, positively autoregu- lates its cognate promoter in both T lymphocytes and in neural cells. This, taken togetherwith the demonstra- ble presence of IE62 in the VZV virion tegument pro- vides a plausible mechanism by which the triggering of gene expression after infection occurs in the absence of a functional VZV a-TIF protein.

REGULATION OF VARICELLA-ZOSTER VIRUS GENE EXPRESSION 353

ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service Grants All8449 (W.T.R. and J.H.; N.I.A.I.D.) and lR29-A124489 (J.D.M.; N.I.A.I.D.). We acknowledge the award of a grant to Dr. Peter Shel- drick, CNRS, Villejuif, France, from I’Association pour la recherche sur ie Cancer in support of M.S-Z.The opinions or assertions con- tained within are private views of the authors and should not be construed as official or necessarily reflecting the views of the Uni- formed Services University of the Health Sciences or the Department of Defense.

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