Virus Research, 15 (1990) 51-68
57
Elsevier VIRUS 00545
Trans-activation of viral tk promoters by proteins encoded by varicella zoster virus open reading frames 61 and 62 Gary F. Cabirac I, Ravi Mahalingam*, Mary Wellish 3 and Donald H. Gilden 2 ’ Rocky Mountain Multiple Sclerosis Englouood CO 80150-0101, U.S.A. and Colorado, Denver, CO 80262, U.S.A. Science
Centers, Colorado Neuroscience Institute, Department ?5OOLB, Department of Biochemistry, Biophysics and Genetics, University of and 2 Department of NeuroIogy, University of Colorado, Health Center, Denver, CO gO262, U.S.A.
(Accepted 22 September 1989)
Plasmids ~nt~~n~ the varicella zoster virus (VZV) open reading frames (ORFs) 61 and 62 were used in a transient co-transfection assay to test for trans-activation of the VZV and herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) promoters. The trams-activating potential of the polypeptides encoded by these VZV ORFs, designated ~51 and ~140, was compared to that of their HSV-1 homologs ICPO and ICP4, respectively. VZV ~51 was functionally inactive in this system while ~140 appeared to be a much stronger transcriptional activator than ICP4. Co-transfection of plasmids encoding VZV ~140 and HSV-1 ICPO resulted in a synergistic activation of the reporter gene as has been shown for the combination of ICP4 and ICPO. Varicella zoster virus; Transcriptional
activation; Immediate early protein
Transcriptional regulation of herpes virus genes during lytic infection is mediated, in conjunction with host factors, by a set of viral polypeptides classified as the
Correspondence
to: G.F. Cabirac, off.
0168-1702/90/$03.50 0 1990 Elsevker Science Publishers B.V. (Biom~c~
Division)
5x
immediate early (IE) or alpha proteins (Honess and Roizman, 1974; Clements et al., 1977; Jones and Roizman, 1979). Viral mutant analysis (Preston, 1979; Dixon and Schaffer, 1980; Sears et al., 1985; Nazos-Mavromara et al., 1986; Sacks et al., 1986; Stow and Stow, 1986; DeLuca and Schaffer, 1987,1988; Sacks and Schaffer, 1987), transient transfection assays (Everett, 1984; Gelman and Silverstein, 1985, 1986, 1987; O’Hare and Hayward, 1985a, b; Quinlan and Knipe, 1985; Everett, 1986, 1987; Shapira et al., 1986; O’Hare and Hayward, 1987) and in vitro transcription assays (Beard et al., 1986; Pizer et al., 1986) have been used to demonstrate the transcriptional stimulation and repression activity of some of these IE proteins. The most thoroug~y characterized IE proteins are the five expressed by herpes simplex virus type 1 (HSV-1). Two of these HSV-1 IE proteins, ICPO and ICP4, function independently as strong transactivators of viral gene transcription and act synergistically in stimulating transcription (Everett, 1984; Gelman and Silverstein, 1985; O’Hare and Hayward, 1985a; Quinlan and Knipe, 1985). ICP4 also represses transcription from its own promoter (Dixon and Schaffer, 1980; DeLuca and Schaffer, 1985; Detuca et al., 1985; O’Hare and Hayward, 1985b). The accumulated data indicate that these two IE proteins are important regulators of virus replication. The varicella zoster virus (VZV) homologs of the HSV-1 IE proteins have been identified from the VZV genome sequence (Davison and Scott, 1986). The inferred amino acid sequence of VZV open reading frames (ORFs) 61 and 62 indicates sequence similarity with HSV-1 ICPO and ICP4, respectively (Davison and Scott, 1986). Transcriptional mapping of the VZV genome (Reinhold et al., 1988) shows that mRNAs are transcribed from the 61 and 62 ORF regions. The molecular weights of two of the four viral proteins expressed at immediate early times of VZV infection (Shiraki and Hyman, 1987) correspond approximately to the predicted sizes for these VZV ORFs, namely 51 kDa for ORF 61 (~51) and 140 kDa for ORF 62 (~140). The amino acid sequence similarity of VZV ~51 with HSV-1 ICPO is limited to the putative zinc finger regions in the amino termini of both polypeptides (Perry et al., 1986). The remainder of ~51 bears no detectable significant regions of sequence similarity to ICPO, and the ~51 polypeptide is approximately 300 amino acids shorter than ICPO. VZV ~140 and HSV-1 ICP4 have considerable amino acid sequence similarity. Comparison of these two polypeptides reveals highly conserved regions with an overall sequence similarity of approximately 50%. (McGeoch et al., 1986). ~140 can act as a trans-activator of transcription in transient transfection assays (Everett, 1984) and can also complement an ICP4 deficient HSV-1 mutant (Felser et al., 1988). Since the herpes virus IE proteins are an important determinant of virus replication we chose to investigate the potential roles that VZV p51 and ~140 play in the unique biology of VZV infection. As a first step we have compared VZV p51 and ~140 to HSV-1 ICPO and ICP4 by using a transient transfection system to test for trans-activation of the VZV and HSV-1 tk promoters.
59
Materials
and Methods
Plasmids
pZ388 contains a 2.9 kb HindIII-SmaI fragment containing the promoter and coding sequences of VZV ~51 ligated into pIBI31 (International Biotechnologies, Inc., pIBI31 contains a multiple cloning site flanked by the T3 and T7 RNA polymerase promoters). The insert in this plasmid extends from 102,110 to 105,018bp in the VZV genome (Davison and Scott, 1986). The SmaI site of the VZV insert is 534 bp upstream from the ORF 61 initiation codon. pN148 contains a 5.0 kb N&I restriction fragment, 104,421 to 109,465 bp of the VZV genome, coding for ~140 inserted into pGEMSZf+ (Promega). The N&I site of this fragment is 336 bp upstream from the ORF 62 initiation codon. plll contains a 4.6 kb SstI-HpaI fragment with the promoter and coding regions of HSV-1 ICPO (Everett, 1987). pJB33, HSV-1 ICP4 promoter and coding sequences, contains an 8.1 kb HindIII-BstEII partial restriction fragment with Hind111 linkers derived from pGX58 (Everett, 1984) ligated into the Hind111 site of pSVOd (Mellon et al., 1981). pSVOd contains SV40 ori sequences and the early transcription start site but lacks enhancer regions. The promoter regions for the VZV and HSV-1 tk genes (described in Results and Fig. l), were cloned into pXP1 (Nordeen, 1988) and designated pZkLuc and pHkLuc, respectively. pXP1, a derivative of pSV232AL-A 5’ (DeWet et al., 1987), contains the coding sequences for the firefly (Photinus pyralis) luciferase gene plus SV40 polyadenylation and splicing signals. All plasmids were propagated in HBlOl, purified by the alkaline-SDS method (Bimboim and Doly, 1979) then purified on CsCl-ethidium bromide gradients. Transfections and luciferase assays CV-1 and HeLa cells were grown in Dulbecco’s Modified Eagle (DME) medium supplemented with 50 pg/rnl gentamicin (Gibco) and 10% fetal bovine serum (FBS) (DME + 10). Cells were seeded at 1 X 105/well into 6-well cluster plates (Costar) approximately 24 h before transfection. Cells were transfected by the DEAE dextran method (Luthman and Magnusson, 1983; Sussman and Milman, 1984) as follows. Plasmid DNAs, dissolved in 1 mM Tris, pH 8.0, 0.1 mM EDTA (0.1 X TE), were added to DME + 10 containing 100 I_IM chloroquine. The concentration of the tk-luciferase plasmid in all experiments was 0.5 pg/rnl and all other plasmids were added in equal molar concentrations; the final DNA concentration was adjusted to 2.0 pg/ml with pUC19 DNA. DEAE dextran was added to a final concentration of 100 (CV-1 cells) or 200 pg/ml (HeLa cells). The DNA-DEAE dextran solution (0.6 ml/35 mm well) was incubated at 37°C for 1 h on CV-1 or 2 h on HeLa cells. Cell monolayers were then washed once with DME + 10, shocked for 2 min with 15% glycerol, Hepes-buffered saline (HBS), washed twice with DME + 10, and incubated at 37” C in fresh medium until harvesting.
60
+1 *
OCTA
.
., f,.$
i
:il
f
I OCTA
TATA’““”
OCTA
G-RICH -210
I PVUII
VZV tk
I G-RICH
CCAAT
I
II CCAAT
SPl
+55
HSV tk
I TATA
Bglll
SPl
Fig. 1. Construction of viral tk promoter-luciferase plasmids. The plasmid pXP1 (Nordeen, 1988) contains the coding region for the firefly (Phorinw pyralis) luciferase gene (light shading) with SV40 (dark shading) and pBR322 sequences (black) with a polylinker upstream of the luciferase coding region. SV40 polyadenylation signals (A,) are shown upstream and dowstream from the luciferase segment. The indicated fragments containing the VZV or HSV tk promoter regions were inserted upstream of the luciferase coding segment and these plasmid constructs were designated pZkLuc and pHkLuc, respectively. Transcription start sites are shown with arrows. Putative regulatory sequences within the VZV promoter fragment are shown by gray boxes to indicate tentative identification (see text). Well characterized HSV promoter elements are shown by black boxes.
Transfected cells were harvested 48 h post-transfection. Monolayers were washed three times with cold calcium/magnesium-free phosphate-buffered saline (PBS) and lysed by the addition of 0.12 ml/well of ice-cold lysis buffer (100 mM KPO,, pH 7.8, 1 mM DTT, 0.5% NP-40). After 3 min, the buffer was removed; nuclei remained attached to the plate. Luciferase levels in these protein extracts were assayed using a Monolight 2001 Luminometer (Analytical Luminescence Laboratory) immediately after preparation of the extracts. The extract (40 ~1 containing 70-120 pg protein as determined by BioRad assay) was mixed with 0.35 ml of 25 mM KPO,, pH 7.8,15 mM MgCl, and 5 mM ATP and light output was measured for 10 s after the injection of 100 ~1 of 1 mM luciferin (Analytical Luminescence). Luciferase activity from each extract was measured in duplicate. Values were normalized to 100 pg protein. Luciferase levels were obtained from a minimum of three separate transfection experiments. DNA was routinely extracted from nuclei after NP-40 lysis of monolayers, digested with appropriate restriction enzymes and
61
analyzed by Southern blot hybridization to insure that there were no significant differences in efficiencies of transfection, Luciferase activity from cells co-transfected with the promoterless reporter plasmid pXP1 and the various tram+activator plasmids was always less than 1% of the activity obtained from co-transfections with the tk-promoter reporter plasmids.
Results Construction of tk-luciferase reporter plasmids
The promoter regions of the VZV and HSV-1 tk genes used in this study are shown in Fig. 1. The transcriptional start site for the VZV tk mRNA has previously been determined (Davison and Scott, 1986). The PuuII site at the + 1 position was chosen since there were no convenient restriction sites between the + 1 position and the translation initiation codon of the gene. While some viral genes require sequences downstream of the cap site for fuII transcriptional activity (Ayer and Dynan, 1988), the construct of the VZV tk promoter used here was sufficient for expression of the luciferase reporter gene. However, the level of expression from this promoter was significantly lower than that from the HSV-1 tk promoter (discussed below). The HSV-1 tk promoter region used here included the octanucleotide motif at position -130 (Bergman et al., 1984; FaIkner and Zachau, 1984; Perry et al., 1985; Sive and Roeder, 1986). One report has implicated this octanucleotide motif in regulation of the HSV-1 tk promoter (Parslow et al., 1987). However, there was no significant difference in the levels of luciferase expression in the cell types used here when this HSV-1 tk promoter construct was compared to a truncated promoter not containing the octanucleotide sequence (data not shown). The regulatory sequences illustrated for the VZV tk promoter were identified on the basis of sequence identity alone. The TATA box region has been identified previously (Davison and Scott, 1986) based on its sequence similarity to other TATA elements and its position relative to the transcriptional start site. The two tentative octanucleotide motifs at - 139 and - 227 have the sequences GTITCCAT and TT’ITGCAT, respectively. Both of these are similar to the canonical octamer sequence of A’MTGCAT (Falkner and Zachau, 1984; Perry et al., 1985). The sequence at - 170, CCAAAT, and flanking regions resemble the many CCAAT-box regulatory elements shown to bind a number of transcription factors (Dorn et al., 1987; Jones et al., 1987; Wingender, 1988). The two G-rich regions at -64 and - 191 may be SPl binding sites or represent some vestigial remnants of such sites (Wingender, 1988). Note that except for the octanucleotide sequence downstream from the CCAAT-box, the relative positions of the tentative regulatory elements in the VZV promoter are the same as those in the HSV-1 promoter. None of these putative VZV tk promoter regulatory elements have been shown, either by functional mutational analysis or transcription factor binding assays, to be important for the transcriptional regulation of the VZV tk gene. The elements were identified to
62
determine the degree of similarity between the org~ization and HSV tk promoter. Tram-actiuation
of the tk promoters
of the VZV tk promoter
in CV-1 cells
Fig. 2 shows the levels of luciferase expression from the tk reporter plasmids in CV-1 cells after DEAE dextran co-transfection with plasmids encoding the various TRANS-ACTIVATION OF TK PROMOTERS IN CVI CELLS
BOO ,
600 FOLD
I
v2v
i
INDUCTION
400
0 4
140
0
51
CO-fF?ANSFECTING
_--
4+0
140+51
4+51
PLASMIDS
I
I
140+0
t--&
I
HSV
200 FOLD INDUCTION
100
.
‘2
II I
x o-r71
c-7
r. 4
140
0
51
CO-TRANSFECTING
4+0
140+51
4+51
140+0
PLASMIDS
Fig. 2. Trans-activation of transcription from the VZV and HSV tk promoters in CV-1 cells. pZkLuc (top) or pHkLuc (bottom) were co-transfected using DEAE dextran with plasmids encoding VZV ~140 (140) and ~51 (51) and HSV-1 ICP4 (4) and ICPO (0) as described in Materials and Methods. Luciferase activities are shown relative to those obtained by transfection of the reporter plasmid alone. Results were obtained from a minimum of three separate transfections. Bars indicate SD of the mean.
63
VZV and HSV-1 IE proteins. The values shown in the graphs represent the fold induction in luciferase activity compared to that obtained after transfection of the reporter plasmid alone. The luciferase activity from cells transfected with the HSV reporter plasmid alone averaged 1980 (arbitrary light units) per 100 pg of extract while activity from cells transfected with the VZV reporter plasmid averaged 50. Activity from cells co-transfected with the HSV-1 reporter plasmid and plasmids encoding VZV ~140 and HSV-1 ICPO averaged 563 100 while activity from cells co-transfected with the VZV reporter plasmid and ~140 and ICPO averaged 34400. Thus, the extent of induction of the VZV tk promoter by the various viral trans-activators was much higher than the extent of induction of the HSV-1 tk promoter. However, the absolute luciferase levels obtained with the HSV-1 tk plasmid were much higher than those from the VZV tk plasmid. Qualitatively, the degree of transcriptional activation by the various VZV and HSV-1 IE products was similar for both viral tk promoters (Fig. 2). However, VZV ~140 was a stronger activator than ICPO of the VZV tk promoter (123-fold vs 64-fold induction) while ICPO was stronger than ~140 when the HSV-1 tk promoter was the target (35-fold vs 18-fold induction). Both the VZV and HSV-1 tk promoters were used here to determine if the pattern of trans-activation by the VZV or HSV-1 proteins would be dependent on cis acting sequences unique to one promoter. Except in the case of ~140 and ICPO mentioned above, there does not seem to be a virus type specific activation of the two promoters. Fig. 2 shows that VZV ~140 appeared to be a stronger trans-activator than ICP4, that it acted synergistically with HSV-1 ICPO and that VZV p51 did not activate either viral tk promoter. The levels of induction by ~140 and ICP4 for the VZV or HSV tk promoters were 123 and 25 or 18 and 7, respectively. Synergism between HSV-1 ICP4 and ICPO has been demonstrated before (Everett, 1984; Gelman and Silverstein, 1985; O’Hare and Hayward, 1985a; Quinlan and Knipe, 1985) but the level of tram-activation by ~140 plus ICPO reported here was greater than the combination of the two HSV-1 proteins. The fact that no activation of the tk promoters is observed with co-transfection of the p51 could simply mean that a mutation exists in this particular p51 plasmid that affects the transcription of the p51 mRNA or the production of a protein. However, Northern blot analysis of RNA isolated from cells transfected with the p51 plasmid shows that a transcript is produced from this plasmid and that this transcript is identical in size to a species detected in VZV infected cells (data not shown). Additionally, other investigators using independently isolated plasmids and different target promoters have found no trans-activation with p51 (R. Everett, J. Ostrove, personal communications). Dependence
of transcriptional
activation
on cell type
Fig. 3 shows the relative levels of luciferase produced after DEAE dextran co-transfection of the HSV-1 tk reporter plasmid and the various trans-activator plasmids into HeLa cells. Compared to CV-1 cells, the relative levels of activation were much lower and there was a qualitative difference in these levels. Co-transfection of HeLa cells with the reporter plasmid and ICP4, ~51, ~140 + ~51, or
64
HSVl
TRANS-ACTIVATION OF TK PROMOTER IN HeLa CELLS
30
-.5, 20 FOLD INDUCTION
0
4
140
0
51
CO-TRANSFECTtNG
4+0
MO+51
4+51
140+0
PLASMIDS
Fig. 3. Transcriptional activation of the HSV-1 tk promoter in HeLa cells by viral trans-activators. pHkLuc and plasmids encoding the VZV and HSV-1 trans-activators were co-transfected using DEAE dextran. The values shown represent the fold-induction of luciferase activity compared to levels obtained from transfection of the reporter ptasmid alone.
ICP4 + ~51 resulted in no significant activation above the basal level (transfection of the reporter plasmid alone). Co-transfection of the reporter plasmid with ~140 and ICPO produced a 2- and 7-fold increase, respectively, in luciferase levels. These two trans-activators produced 1% and 3%fold inductions, respectively, in CV-1 cells. The synergism seen between ICP4 and ICPO in CV-1 cells was not observed in the HeLa cells. Rather, the combination of these two trans-activators produced a lower level of activation than ICPO alone (2.5fold vs 7-fold). However, the combination of ~140 plus ICPO still had a synergistic effect on promoter activation producing a 24-fold increase in luciferase activity. Transfection of the VZV tk reporter plasmid into HeLa cells either alone or co-transfected with most of the trans-activator plasmids did not produce detectable luciferase activity. Only cotransfection of the VZV tk reporter plasmid with ~140 and ICPO into HeLa cells resulted in measurable amounts of luciferase activity (data not shown).
Discussion
Transcriptional activation by two putative immediate early VZV proteins, ~140 and ~51, was compared to that by HSV-1 ICP4 and ICPO using both VZV and HSV tk promoters as targets of trans-activation. VZV ~140 appears to be a stronger trans-activator than HSV-1 ICP4, and acts synergistically with HSV-1 ICPO. VZV p51 did not tram-activate the target promoters.
65
In comparing trans-activation by VZV ~140 and HSV-1 ICP4 the autoregulatory function of the ICP4 protein needs to be considered. (Dixon and Schaffer, 1980; DeLuca and Schaffer, 1985; DeLuca et al., 1985; O’Hare and Hayward, 1985b). A recent report describing stably transfected cell lines with VZV ~140 provides evidence that this viral protein may also be autoregulatory (Felser et al., 1988). Our preliminary transfection experiments suggest that ~140 may not repress its own promoter. However, even if ~140 is autoregulatory, a more accurate comparison of tram-activation strengths would be obtained using equivalent non-repressible promoter to drive expression of ~140 and ICP4. Detailed mutational analysis of the ICPO pol~eptide (Everett, 1987, 1988) has shown that this viral trans-activator has multiple functional regions. Since there is only limited sequence similarity between p51 and ICPO it is hard to predict why ~51 lacks trans-activation function. However, ~51 could trans-activate a specific subset of promoters or acquire a transcriptional activation function upon interaction with another viral protein(s). Therefore our results do not eliminate the possibility that ~51 functions as a trans-activator in vivo or that it plays another role during virus replication. The apparent lack of a transcriptional activation function by p51 may be an important factor in the replication of VZV. Studies with HSV-1 containing deletions in both copies of the ICPO gene have shown that loss of this gene results in impaired growth in culture (Stow and Stow, 1986; Sacks and Schaffer, 1987). One of these ICPO deletion mutants was also used in an in vitro latency system to demonstrate that a functional ICY0 product is required for reactivation of virus from the latent state (Russell et al., 1987). Considering these findings, it is tempting to speculate that the poor replication of VZV in vitro and/or the low incidence of reactivation of this virus in vivo may be due in part to the lack of a functional ICPO homolog. Obviously many factors are important in deterring the overall level of virus replication but the relationship between the strength of a trans-activator and virus growth can be important. This has been demonstrated with ElA and adenovirus types 40 and 41 (Van Loon et al., 1987). Further characterization of the VZV IE proteins and comparison of such data to the accumulated information on HSV IE protein function may help explain some of the differences in replication between VZV and HSV. We are currently screening for other VZV transcriptional regulatory proteins and testing the effects of these proteins on virus growth. Since herpesvirus replication is primarily controlled at the level of transcription this line of investigation may provide insight into the unique biology of VZV.
Acknowkdgements
We thank Drs Roger Everett and Joan Betz for the HSV IE plasmids and Dr Peter Sarnow for the CV-1 and HeLa cell lines. We are especially grateful to Dr Steve Nordeen for the pXP luciferase plasmids and advice on the luciferase assays. We appreciate the helpful discussions and critical reading of the manuscript by Joan
66
Betz and Dave Tedder. This work was supported in part by Public Health Service Grants AG-06127 and AG-07347 from the National Institute of Aging. G.F.C. is the recipient of fellowship award GM1167502 from the National Institute of General Medical Science.
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Gelman, I.H. and Silverstein, S. (1985) Identification of immediate-early genes from herpes simplex virus that transactivate the virus thymidine kinase gene. Prw. Natl. Acad. Sci. USA 82,5265-5269. Gelman, I.H. and Silverstein, S. (1986) Co-ordinate regulation of herpes simplex virus gene expression is mediated by the functional interaction of two immediate early gene products. J. Mol. Biol. 191, 395-409. Gelman, I.H. and Silverstein, S. (1987) Dissection of immediate-early gene promoters from herpes simplex virus: sequences that respond to the virus transcriptional activators. J. Virol. 61, 3167-3172. Honess, R.W. and Roizman, B. (1974) Regulation of herpesvirus macromolecular synthesis. 1. Cascade regulation of the synthesis of three groups of viral proteins. J. Virol. 14, 8-19. Jones, K.A., Kadonaga, J.T., Rosenfeld, P.J., Kelly, T.J. and Tjian, R. (1987) A cellular DNA-binding protein that activates eukaryotic tr~~~ption and DNA replication. Cell 48, 79-89. Jones, P.C. and Roizman, B. (1979) Regulation of herpesvirus macromolecular synthesis. VIII. The transcription program consists of three phases during whikh both extent of transcription and accumulation of RNA in the cytoplasm are regulated. J. Virol. 31, 299-314. Luthman, H. and Magnusson, G. (1983) High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res. 11, 1295-1308. McGeoch, D.J., Dolan, A., Donald, S. and Brauer, D.H.K. (1986) Complete DNA sequence of the short repeat region in the genome of herpes simplex virus type 1. Nucleic Acids Res. 14, 1727-1745. Mellon, P., Parker, V., Gluzman, Y. and Maniatis, T. (1981) Identification of DNA sequences required for transcription of the human l-globin gene in a new SV40 host-vector system. Cell 27, 279-288. Nazos-Mavromara, P., Ackermann, M. and Roizman, B. (1986) Construction and properties of a viable herpes simplex virus 1 recombinant lacking coding sequences of the alpha47 gene. J. Virol. 60, 807-812. Nordeen, S. (1988) Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques 6, 454-456. O’Hare, P. and Hayward, G.S. (1985a) Evidence for a direct role for both the 175,ooO and 110,000 molecular weight immediate early proteins of herpes simplex virus in the transactivation of delayed early promoters. J. Virol. 53, 751-760. O’Hare, P. and Hayward, G.S. (1985b) Three trans-acting regulatory proteins of herpes simplex virus modulate immediate-early gene expression in a pathway involving positive and negative feedback regulation. J. Virol. 56, 723-733. O’Hare, P. and Hayward, G.S. (1987) Comparison of upstream sequence requirements for positive and negative regulation of a herpes simplex virus immediate-early gene by three virus-encoded tram-acting factors. J. Virol. 61, 190-199. Parslow, T.G., Jones, D.S., Bond, B. and Yamamoto, K.R. (1987) The immunoglobulin octatmcleotide: independent activity and selective interaction with enhancers. Science 235, 1498-1500. Perry, L.J., Rixon, F.J., Everett, R.D., Frame, MC. and McGeoch, D.J. (1986) Characterization of the IEllO gene of herpes simplex virus type 1. J. Gen. Viral. 67, 2365-2380. Perry, M., Tbomsen, G.H. and Roeder, R.G. (1985) Genomic organization and nucleotide sequence of two distinct histone gene clusters from X&opus lueuis. J. Mol. Biol. 185, 479-499. Pizer, L.I., Tedder, D.G., Betz, J.L., Wilcox, K.W. and Beard, P. (1986) Regulation of transcription in vitro from herpes simplex virus genes. J. Virol. 60, 950-959. Preston, C.M. (1979) Control of herpes simplex virus type 1 mRNA synthesis in cells infected with wild-type virus or the temperature-sensitive mutant tsK. J. Virol. 29, 275-284. Quinlan, M. and Knipe, D. (1985) Stimulation of expression of a herpes simplex virus DNA binding protein by two viral functions. Mol. Cell. Biol. 5, 957-963. Reinhold, W.C., Straus, S.E. and Ostrove, J.M. (1988) Directionality and further mapping of varicella zoster virus transcripts. Virus Res. 9, 249-261. Russell, J., Stow, N.D., Stow, E.C. and Preston, CM. (1987) Herpes simplex virus genes involved in latency in vitro. J. Gen. Virol. 68, 3009-3018. Sacks, W.R., Greene, C.C., As&man, D.P. and Schaffer, P.A. (1986) Herpes simplex virus type I ICP27 is an essential regulatory protein. J. Virol. 55, 796-805, Sacks, W.R. and Schaffer, P.A. (1987) Deletion mutants in the gene encoding the herpes simplex virus type 1 immediate-early protein ICPO exhibit impaired growth in cell culture. J. Virol. 61, 829-839.
68 Sears, A.E., Halliburton, I.W., Meignier, B., Silver, S. and Roizman, B. (1985) Herpes simplex virus 1 mutant deleted in the 22 gene: growth and gene expression in permissive and restrictive cells and establishment of latency in mice. J. Virol. 55, 338-346. Shapira, M., Homa, F.L., Glorioso, J.C. and Levine, M. (1986) Regulation of the herpes simplex virus type 1 late glycoprotein C gene: sequences between base pairs -34 to +29 control transient expression and responsiveness to transactivation by the products of the immediate early 4 and 0 genes. Nucleic Acids Res. 15, 3097-3111. Shiraki, K. and Hyman, R.W. (1987) The immediate early proteins of varicella-zoster virus. Virology 156, 423-426. Sive, H.L. and Roeder, A.G. (1986) Interaction of a common factor with conserved promoter and enhancer sequences in histone H2B, immunoglobulin and U2 small nuclear RNA (snRNA) genes. Proc. Natl. Acad. Sci. USA 83, 6382-6386. Stow, N.D. and Stow, E.C. (1986) Isolation and characterization of a herpes simplex virus type 1 mutant containing a deletion within the gene encoding the immediate early polypeptide VmwllO. J. Gen. Virol. 67, 2571-2587. Sussman, D.J. and Milman, G. (1984) Short term, high efficiency expression of transfected DNA. Mol. Cell. Biol. 4, 1641-1643. Van Loon, A.E., Gilardi, P., Penicaudet, M., Rozun, T.H.H. and Bach, S.N. (1987) Transcriptional activation by the ElA regions of adenovirus types 40 and 41. Virology 160, 305-307. Wingender, E. (1988) Compilation of transcription regulating proteins. Nucleic Acids Res. 16,1879-1902. (Received
6 May 1989; revision
received
19 September
1989)
57
Elsevier VIRUS 00545
Trans-activation of viral tk promoters by proteins encoded by varicella zoster virus open reading frames 61 and 62 Gary F. Cabirac I, Ravi Mahalingam*, Mary Wellish 3 and Donald H. Gilden 2 ’ Rocky Mountain Multiple Sclerosis Englouood CO 80150-0101, U.S.A. and Colorado, Denver, CO 80262, U.S.A. Science
Centers, Colorado Neuroscience Institute, Department ?5OOLB, Department of Biochemistry, Biophysics and Genetics, University of and 2 Department of NeuroIogy, University of Colorado, Health Center, Denver, CO gO262, U.S.A.
(Accepted 22 September 1989)
Plasmids ~nt~~n~ the varicella zoster virus (VZV) open reading frames (ORFs) 61 and 62 were used in a transient co-transfection assay to test for trans-activation of the VZV and herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) promoters. The trams-activating potential of the polypeptides encoded by these VZV ORFs, designated ~51 and ~140, was compared to that of their HSV-1 homologs ICPO and ICP4, respectively. VZV ~51 was functionally inactive in this system while ~140 appeared to be a much stronger transcriptional activator than ICP4. Co-transfection of plasmids encoding VZV ~140 and HSV-1 ICPO resulted in a synergistic activation of the reporter gene as has been shown for the combination of ICP4 and ICPO. Varicella zoster virus; Transcriptional
activation; Immediate early protein
Transcriptional regulation of herpes virus genes during lytic infection is mediated, in conjunction with host factors, by a set of viral polypeptides classified as the
Correspondence
to: G.F. Cabirac, off.
0168-1702/90/$03.50 0 1990 Elsevker Science Publishers B.V. (Biom~c~
Division)
5x
immediate early (IE) or alpha proteins (Honess and Roizman, 1974; Clements et al., 1977; Jones and Roizman, 1979). Viral mutant analysis (Preston, 1979; Dixon and Schaffer, 1980; Sears et al., 1985; Nazos-Mavromara et al., 1986; Sacks et al., 1986; Stow and Stow, 1986; DeLuca and Schaffer, 1987,1988; Sacks and Schaffer, 1987), transient transfection assays (Everett, 1984; Gelman and Silverstein, 1985, 1986, 1987; O’Hare and Hayward, 1985a, b; Quinlan and Knipe, 1985; Everett, 1986, 1987; Shapira et al., 1986; O’Hare and Hayward, 1987) and in vitro transcription assays (Beard et al., 1986; Pizer et al., 1986) have been used to demonstrate the transcriptional stimulation and repression activity of some of these IE proteins. The most thoroug~y characterized IE proteins are the five expressed by herpes simplex virus type 1 (HSV-1). Two of these HSV-1 IE proteins, ICPO and ICP4, function independently as strong transactivators of viral gene transcription and act synergistically in stimulating transcription (Everett, 1984; Gelman and Silverstein, 1985; O’Hare and Hayward, 1985a; Quinlan and Knipe, 1985). ICP4 also represses transcription from its own promoter (Dixon and Schaffer, 1980; DeLuca and Schaffer, 1985; Detuca et al., 1985; O’Hare and Hayward, 1985b). The accumulated data indicate that these two IE proteins are important regulators of virus replication. The varicella zoster virus (VZV) homologs of the HSV-1 IE proteins have been identified from the VZV genome sequence (Davison and Scott, 1986). The inferred amino acid sequence of VZV open reading frames (ORFs) 61 and 62 indicates sequence similarity with HSV-1 ICPO and ICP4, respectively (Davison and Scott, 1986). Transcriptional mapping of the VZV genome (Reinhold et al., 1988) shows that mRNAs are transcribed from the 61 and 62 ORF regions. The molecular weights of two of the four viral proteins expressed at immediate early times of VZV infection (Shiraki and Hyman, 1987) correspond approximately to the predicted sizes for these VZV ORFs, namely 51 kDa for ORF 61 (~51) and 140 kDa for ORF 62 (~140). The amino acid sequence similarity of VZV ~51 with HSV-1 ICPO is limited to the putative zinc finger regions in the amino termini of both polypeptides (Perry et al., 1986). The remainder of ~51 bears no detectable significant regions of sequence similarity to ICPO, and the ~51 polypeptide is approximately 300 amino acids shorter than ICPO. VZV ~140 and HSV-1 ICP4 have considerable amino acid sequence similarity. Comparison of these two polypeptides reveals highly conserved regions with an overall sequence similarity of approximately 50%. (McGeoch et al., 1986). ~140 can act as a trans-activator of transcription in transient transfection assays (Everett, 1984) and can also complement an ICP4 deficient HSV-1 mutant (Felser et al., 1988). Since the herpes virus IE proteins are an important determinant of virus replication we chose to investigate the potential roles that VZV p51 and ~140 play in the unique biology of VZV infection. As a first step we have compared VZV p51 and ~140 to HSV-1 ICPO and ICP4 by using a transient transfection system to test for trans-activation of the VZV and HSV-1 tk promoters.
59
Materials
and Methods
Plasmids
pZ388 contains a 2.9 kb HindIII-SmaI fragment containing the promoter and coding sequences of VZV ~51 ligated into pIBI31 (International Biotechnologies, Inc., pIBI31 contains a multiple cloning site flanked by the T3 and T7 RNA polymerase promoters). The insert in this plasmid extends from 102,110 to 105,018bp in the VZV genome (Davison and Scott, 1986). The SmaI site of the VZV insert is 534 bp upstream from the ORF 61 initiation codon. pN148 contains a 5.0 kb N&I restriction fragment, 104,421 to 109,465 bp of the VZV genome, coding for ~140 inserted into pGEMSZf+ (Promega). The N&I site of this fragment is 336 bp upstream from the ORF 62 initiation codon. plll contains a 4.6 kb SstI-HpaI fragment with the promoter and coding regions of HSV-1 ICPO (Everett, 1987). pJB33, HSV-1 ICP4 promoter and coding sequences, contains an 8.1 kb HindIII-BstEII partial restriction fragment with Hind111 linkers derived from pGX58 (Everett, 1984) ligated into the Hind111 site of pSVOd (Mellon et al., 1981). pSVOd contains SV40 ori sequences and the early transcription start site but lacks enhancer regions. The promoter regions for the VZV and HSV-1 tk genes (described in Results and Fig. l), were cloned into pXP1 (Nordeen, 1988) and designated pZkLuc and pHkLuc, respectively. pXP1, a derivative of pSV232AL-A 5’ (DeWet et al., 1987), contains the coding sequences for the firefly (Photinus pyralis) luciferase gene plus SV40 polyadenylation and splicing signals. All plasmids were propagated in HBlOl, purified by the alkaline-SDS method (Bimboim and Doly, 1979) then purified on CsCl-ethidium bromide gradients. Transfections and luciferase assays CV-1 and HeLa cells were grown in Dulbecco’s Modified Eagle (DME) medium supplemented with 50 pg/rnl gentamicin (Gibco) and 10% fetal bovine serum (FBS) (DME + 10). Cells were seeded at 1 X 105/well into 6-well cluster plates (Costar) approximately 24 h before transfection. Cells were transfected by the DEAE dextran method (Luthman and Magnusson, 1983; Sussman and Milman, 1984) as follows. Plasmid DNAs, dissolved in 1 mM Tris, pH 8.0, 0.1 mM EDTA (0.1 X TE), were added to DME + 10 containing 100 I_IM chloroquine. The concentration of the tk-luciferase plasmid in all experiments was 0.5 pg/rnl and all other plasmids were added in equal molar concentrations; the final DNA concentration was adjusted to 2.0 pg/ml with pUC19 DNA. DEAE dextran was added to a final concentration of 100 (CV-1 cells) or 200 pg/ml (HeLa cells). The DNA-DEAE dextran solution (0.6 ml/35 mm well) was incubated at 37°C for 1 h on CV-1 or 2 h on HeLa cells. Cell monolayers were then washed once with DME + 10, shocked for 2 min with 15% glycerol, Hepes-buffered saline (HBS), washed twice with DME + 10, and incubated at 37” C in fresh medium until harvesting.
60
+1 *
OCTA
.
., f,.$
i
:il
f
I OCTA
TATA’““”
OCTA
G-RICH -210
I PVUII
VZV tk
I G-RICH
CCAAT
I
II CCAAT
SPl
+55
HSV tk
I TATA
Bglll
SPl
Fig. 1. Construction of viral tk promoter-luciferase plasmids. The plasmid pXP1 (Nordeen, 1988) contains the coding region for the firefly (Phorinw pyralis) luciferase gene (light shading) with SV40 (dark shading) and pBR322 sequences (black) with a polylinker upstream of the luciferase coding region. SV40 polyadenylation signals (A,) are shown upstream and dowstream from the luciferase segment. The indicated fragments containing the VZV or HSV tk promoter regions were inserted upstream of the luciferase coding segment and these plasmid constructs were designated pZkLuc and pHkLuc, respectively. Transcription start sites are shown with arrows. Putative regulatory sequences within the VZV promoter fragment are shown by gray boxes to indicate tentative identification (see text). Well characterized HSV promoter elements are shown by black boxes.
Transfected cells were harvested 48 h post-transfection. Monolayers were washed three times with cold calcium/magnesium-free phosphate-buffered saline (PBS) and lysed by the addition of 0.12 ml/well of ice-cold lysis buffer (100 mM KPO,, pH 7.8, 1 mM DTT, 0.5% NP-40). After 3 min, the buffer was removed; nuclei remained attached to the plate. Luciferase levels in these protein extracts were assayed using a Monolight 2001 Luminometer (Analytical Luminescence Laboratory) immediately after preparation of the extracts. The extract (40 ~1 containing 70-120 pg protein as determined by BioRad assay) was mixed with 0.35 ml of 25 mM KPO,, pH 7.8,15 mM MgCl, and 5 mM ATP and light output was measured for 10 s after the injection of 100 ~1 of 1 mM luciferin (Analytical Luminescence). Luciferase activity from each extract was measured in duplicate. Values were normalized to 100 pg protein. Luciferase levels were obtained from a minimum of three separate transfection experiments. DNA was routinely extracted from nuclei after NP-40 lysis of monolayers, digested with appropriate restriction enzymes and
61
analyzed by Southern blot hybridization to insure that there were no significant differences in efficiencies of transfection, Luciferase activity from cells co-transfected with the promoterless reporter plasmid pXP1 and the various tram+activator plasmids was always less than 1% of the activity obtained from co-transfections with the tk-promoter reporter plasmids.
Results Construction of tk-luciferase reporter plasmids
The promoter regions of the VZV and HSV-1 tk genes used in this study are shown in Fig. 1. The transcriptional start site for the VZV tk mRNA has previously been determined (Davison and Scott, 1986). The PuuII site at the + 1 position was chosen since there were no convenient restriction sites between the + 1 position and the translation initiation codon of the gene. While some viral genes require sequences downstream of the cap site for fuII transcriptional activity (Ayer and Dynan, 1988), the construct of the VZV tk promoter used here was sufficient for expression of the luciferase reporter gene. However, the level of expression from this promoter was significantly lower than that from the HSV-1 tk promoter (discussed below). The HSV-1 tk promoter region used here included the octanucleotide motif at position -130 (Bergman et al., 1984; FaIkner and Zachau, 1984; Perry et al., 1985; Sive and Roeder, 1986). One report has implicated this octanucleotide motif in regulation of the HSV-1 tk promoter (Parslow et al., 1987). However, there was no significant difference in the levels of luciferase expression in the cell types used here when this HSV-1 tk promoter construct was compared to a truncated promoter not containing the octanucleotide sequence (data not shown). The regulatory sequences illustrated for the VZV tk promoter were identified on the basis of sequence identity alone. The TATA box region has been identified previously (Davison and Scott, 1986) based on its sequence similarity to other TATA elements and its position relative to the transcriptional start site. The two tentative octanucleotide motifs at - 139 and - 227 have the sequences GTITCCAT and TT’ITGCAT, respectively. Both of these are similar to the canonical octamer sequence of A’MTGCAT (Falkner and Zachau, 1984; Perry et al., 1985). The sequence at - 170, CCAAAT, and flanking regions resemble the many CCAAT-box regulatory elements shown to bind a number of transcription factors (Dorn et al., 1987; Jones et al., 1987; Wingender, 1988). The two G-rich regions at -64 and - 191 may be SPl binding sites or represent some vestigial remnants of such sites (Wingender, 1988). Note that except for the octanucleotide sequence downstream from the CCAAT-box, the relative positions of the tentative regulatory elements in the VZV promoter are the same as those in the HSV-1 promoter. None of these putative VZV tk promoter regulatory elements have been shown, either by functional mutational analysis or transcription factor binding assays, to be important for the transcriptional regulation of the VZV tk gene. The elements were identified to
62
determine the degree of similarity between the org~ization and HSV tk promoter. Tram-actiuation
of the tk promoters
of the VZV tk promoter
in CV-1 cells
Fig. 2 shows the levels of luciferase expression from the tk reporter plasmids in CV-1 cells after DEAE dextran co-transfection with plasmids encoding the various TRANS-ACTIVATION OF TK PROMOTERS IN CVI CELLS
BOO ,
600 FOLD
I
v2v
i
INDUCTION
400
0 4
140
0
51
CO-fF?ANSFECTING
_--
4+0
140+51
4+51
PLASMIDS
I
I
140+0
t--&
I
HSV
200 FOLD INDUCTION
100
.
‘2
II I
x o-r71
c-7
r. 4
140
0
51
CO-TRANSFECTING
4+0
140+51
4+51
140+0
PLASMIDS
Fig. 2. Trans-activation of transcription from the VZV and HSV tk promoters in CV-1 cells. pZkLuc (top) or pHkLuc (bottom) were co-transfected using DEAE dextran with plasmids encoding VZV ~140 (140) and ~51 (51) and HSV-1 ICP4 (4) and ICPO (0) as described in Materials and Methods. Luciferase activities are shown relative to those obtained by transfection of the reporter plasmid alone. Results were obtained from a minimum of three separate transfections. Bars indicate SD of the mean.
63
VZV and HSV-1 IE proteins. The values shown in the graphs represent the fold induction in luciferase activity compared to that obtained after transfection of the reporter plasmid alone. The luciferase activity from cells transfected with the HSV reporter plasmid alone averaged 1980 (arbitrary light units) per 100 pg of extract while activity from cells transfected with the VZV reporter plasmid averaged 50. Activity from cells co-transfected with the HSV-1 reporter plasmid and plasmids encoding VZV ~140 and HSV-1 ICPO averaged 563 100 while activity from cells co-transfected with the VZV reporter plasmid and ~140 and ICPO averaged 34400. Thus, the extent of induction of the VZV tk promoter by the various viral trans-activators was much higher than the extent of induction of the HSV-1 tk promoter. However, the absolute luciferase levels obtained with the HSV-1 tk plasmid were much higher than those from the VZV tk plasmid. Qualitatively, the degree of transcriptional activation by the various VZV and HSV-1 IE products was similar for both viral tk promoters (Fig. 2). However, VZV ~140 was a stronger activator than ICPO of the VZV tk promoter (123-fold vs 64-fold induction) while ICPO was stronger than ~140 when the HSV-1 tk promoter was the target (35-fold vs 18-fold induction). Both the VZV and HSV-1 tk promoters were used here to determine if the pattern of trans-activation by the VZV or HSV-1 proteins would be dependent on cis acting sequences unique to one promoter. Except in the case of ~140 and ICPO mentioned above, there does not seem to be a virus type specific activation of the two promoters. Fig. 2 shows that VZV ~140 appeared to be a stronger trans-activator than ICP4, that it acted synergistically with HSV-1 ICPO and that VZV p51 did not activate either viral tk promoter. The levels of induction by ~140 and ICP4 for the VZV or HSV tk promoters were 123 and 25 or 18 and 7, respectively. Synergism between HSV-1 ICP4 and ICPO has been demonstrated before (Everett, 1984; Gelman and Silverstein, 1985; O’Hare and Hayward, 1985a; Quinlan and Knipe, 1985) but the level of tram-activation by ~140 plus ICPO reported here was greater than the combination of the two HSV-1 proteins. The fact that no activation of the tk promoters is observed with co-transfection of the p51 could simply mean that a mutation exists in this particular p51 plasmid that affects the transcription of the p51 mRNA or the production of a protein. However, Northern blot analysis of RNA isolated from cells transfected with the p51 plasmid shows that a transcript is produced from this plasmid and that this transcript is identical in size to a species detected in VZV infected cells (data not shown). Additionally, other investigators using independently isolated plasmids and different target promoters have found no trans-activation with p51 (R. Everett, J. Ostrove, personal communications). Dependence
of transcriptional
activation
on cell type
Fig. 3 shows the relative levels of luciferase produced after DEAE dextran co-transfection of the HSV-1 tk reporter plasmid and the various trans-activator plasmids into HeLa cells. Compared to CV-1 cells, the relative levels of activation were much lower and there was a qualitative difference in these levels. Co-transfection of HeLa cells with the reporter plasmid and ICP4, ~51, ~140 + ~51, or
64
HSVl
TRANS-ACTIVATION OF TK PROMOTER IN HeLa CELLS
30
-.5, 20 FOLD INDUCTION
0
4
140
0
51
CO-TRANSFECTtNG
4+0
MO+51
4+51
140+0
PLASMIDS
Fig. 3. Transcriptional activation of the HSV-1 tk promoter in HeLa cells by viral trans-activators. pHkLuc and plasmids encoding the VZV and HSV-1 trans-activators were co-transfected using DEAE dextran. The values shown represent the fold-induction of luciferase activity compared to levels obtained from transfection of the reporter ptasmid alone.
ICP4 + ~51 resulted in no significant activation above the basal level (transfection of the reporter plasmid alone). Co-transfection of the reporter plasmid with ~140 and ICPO produced a 2- and 7-fold increase, respectively, in luciferase levels. These two trans-activators produced 1% and 3%fold inductions, respectively, in CV-1 cells. The synergism seen between ICP4 and ICPO in CV-1 cells was not observed in the HeLa cells. Rather, the combination of these two trans-activators produced a lower level of activation than ICPO alone (2.5fold vs 7-fold). However, the combination of ~140 plus ICPO still had a synergistic effect on promoter activation producing a 24-fold increase in luciferase activity. Transfection of the VZV tk reporter plasmid into HeLa cells either alone or co-transfected with most of the trans-activator plasmids did not produce detectable luciferase activity. Only cotransfection of the VZV tk reporter plasmid with ~140 and ICPO into HeLa cells resulted in measurable amounts of luciferase activity (data not shown).
Discussion
Transcriptional activation by two putative immediate early VZV proteins, ~140 and ~51, was compared to that by HSV-1 ICP4 and ICPO using both VZV and HSV tk promoters as targets of trans-activation. VZV ~140 appears to be a stronger trans-activator than HSV-1 ICP4, and acts synergistically with HSV-1 ICPO. VZV p51 did not tram-activate the target promoters.
65
In comparing trans-activation by VZV ~140 and HSV-1 ICP4 the autoregulatory function of the ICP4 protein needs to be considered. (Dixon and Schaffer, 1980; DeLuca and Schaffer, 1985; DeLuca et al., 1985; O’Hare and Hayward, 1985b). A recent report describing stably transfected cell lines with VZV ~140 provides evidence that this viral protein may also be autoregulatory (Felser et al., 1988). Our preliminary transfection experiments suggest that ~140 may not repress its own promoter. However, even if ~140 is autoregulatory, a more accurate comparison of tram-activation strengths would be obtained using equivalent non-repressible promoter to drive expression of ~140 and ICP4. Detailed mutational analysis of the ICPO pol~eptide (Everett, 1987, 1988) has shown that this viral trans-activator has multiple functional regions. Since there is only limited sequence similarity between p51 and ICPO it is hard to predict why ~51 lacks trans-activation function. However, ~51 could trans-activate a specific subset of promoters or acquire a transcriptional activation function upon interaction with another viral protein(s). Therefore our results do not eliminate the possibility that ~51 functions as a trans-activator in vivo or that it plays another role during virus replication. The apparent lack of a transcriptional activation function by p51 may be an important factor in the replication of VZV. Studies with HSV-1 containing deletions in both copies of the ICPO gene have shown that loss of this gene results in impaired growth in culture (Stow and Stow, 1986; Sacks and Schaffer, 1987). One of these ICPO deletion mutants was also used in an in vitro latency system to demonstrate that a functional ICY0 product is required for reactivation of virus from the latent state (Russell et al., 1987). Considering these findings, it is tempting to speculate that the poor replication of VZV in vitro and/or the low incidence of reactivation of this virus in vivo may be due in part to the lack of a functional ICPO homolog. Obviously many factors are important in deterring the overall level of virus replication but the relationship between the strength of a trans-activator and virus growth can be important. This has been demonstrated with ElA and adenovirus types 40 and 41 (Van Loon et al., 1987). Further characterization of the VZV IE proteins and comparison of such data to the accumulated information on HSV IE protein function may help explain some of the differences in replication between VZV and HSV. We are currently screening for other VZV transcriptional regulatory proteins and testing the effects of these proteins on virus growth. Since herpesvirus replication is primarily controlled at the level of transcription this line of investigation may provide insight into the unique biology of VZV.
Acknowkdgements
We thank Drs Roger Everett and Joan Betz for the HSV IE plasmids and Dr Peter Sarnow for the CV-1 and HeLa cell lines. We are especially grateful to Dr Steve Nordeen for the pXP luciferase plasmids and advice on the luciferase assays. We appreciate the helpful discussions and critical reading of the manuscript by Joan
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Betz and Dave Tedder. This work was supported in part by Public Health Service Grants AG-06127 and AG-07347 from the National Institute of Aging. G.F.C. is the recipient of fellowship award GM1167502 from the National Institute of General Medical Science.
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6 May 1989; revision
received
19 September
1989)
