Varicella-zoster virus (VZV) causes chicken pox (varicella),
becomes latent in dorsal root ganglia, and reactivates decades later to
cause shingles (zoster). During latency, the entire VZV genome is
present in a circular form, from which genes 21, 29, 62, and 63 are
transcribed. Immediate-early (IE) VZV genes 62 and 63 encode regulators
of virus gene transcription, and VZV gene 29 encodes a major
DNA-binding protein. However, little is known about the function of VZV
gene 21 or the control of its transcription. Using primer extensions,
we mapped the start of VZV gene 21 transcription in VZV-infected cells
to a single site located at 
79 nucleotides (nt) with respect to the
initiation codon. To identify the VZV gene 21 promoter, the 284-bp
region of VZV DNA separating open reading frames (ORFs) 20 and 21 was cloned upstream from the chloramphenicol acetyltransferase gene. In
transient-transfection assays, the VZV gene 21 promoter was transactivated in VZV-infected, but not uninfected, cells. Further, the
protein encoded by ORF 62 (IE62), but not those encoded by VZV ORFs 4, 10, 61, and 63, transactivates the VZV gene 21 promoter. By use of
transient-cotransfection assays in conjunction with 5' deletions of the
VZV gene 21 promoter, a 40-bp segment was shown to be responsible for
the transactivation of the VZV gene 21 promoter by IE62. This region
was located at 96 to 56 nt with respect to the 5' start of gene 21 transcription.
 |
INTRODUCTION |
Varicella-zoster virus (VZV), a
neurotropic alphaherpesvirus, causes childhood chicken pox (varicella),
becomes latent in dorsal root ganglia at all levels of the neuraxis,
and may reactivate decades later to produce shingles (zoster)
(18). The entire 125,884-bp VZV genome has been sequenced,
and 71 open reading frames (ORFs) have been identified (14).
Transcripts mapping to most of the predicted ORFs have been detected in
VZV-infected cells, although fewer than 20 VZV genes have been analyzed
in detail (29, 35, 37). The VZV genome is compact: the 71 ORFs are separated by an average of 211 bp, indicating that the
promoters are close to the genes they control.
Coordinated control of virus gene expression is a hallmark of
herpesvirus lytic replication (21). Because VZV is highly cell associated and does not grow to high titers, experiments involving
high multiplicities of infection and single-step virus growth have been
difficult. Nevertheless, it appears that like that of the prototype
alpha-herpesvirus, herpes simplex virus type 1 (HSV-1), VZV gene
transcription during productive infection is highly regulated and
follows a complex cascade of events (8). VZV immediate-early
(IE) genes are the first to be transcribed, and their promoters are
recognized by the existing cellular transcription factors. VZV IE
proteins transactivate promoters for the VZV genes involved in virus
DNA replication (early proteins). During this time, the input linear
herpesvirus DNA circularizes in preparation for replication via a
rolling-circle mechanism (3, 25). Following the initiation
of virus DNA replication, late viral genes are transcribed and
translated. Late gene promoters are not recognized by unmodified
cellular transcription factors, and their transactivation by IE
proteins is only marginal. However, gene amplification resulting from
virus DNA replication overcomes late promoter inefficiency, and late
gene transcripts accumulate in infected cells.
During latency, productive VZV replication is blocked, VZV is not seen
by electron microscopy in otherwise normal human ganglia, and
infectious virus cannot be recovered (17). The VZV genome exists in an episomal form from which at least four virus genes (genes
21, 29, 62, and 63) are transcribed (6, 9, 13). VZV genes 62 and 63 encode IE phosphoproteins which orchestrate virus gene
transcription (2, 15, 16, 24, 26, 27, 36), and VZV gene 29 encodes a 130-kDa early DNA-binding protein (28). The
function of the VZV gene 21 protein has not been studied, although its
HSV-1 homolog, UL37, binds to HSV-1 ICP8, the homolog of the VZV gene
29 protein (39, 40). To begin studying the structure and
regulation of VZV gene 21, we used primer extensions to identify the 5'
start of VZV gene 21 transcription and transient-transfection assays to
locate the promoter for VZV gene 21 in infected cells.
| |
MATERIALS AND METHODS |
Virus and cells.
VZV (Ellen strain) was propagated in a
continuous line of African green monkey kidney cells (BSC-1) in
Dulbecco's minimal essential medium supplemented with 10% fetal calf
serum. Infected cells were cocultivated with uninfected cells as
described previously (19).
RNA extraction and primer extension.
Uninfected and
VZV-infected BSC-1 cells were disrupted with guanidine lysis buffer,
and total RNA was extracted with acid-phenol (5). Table
1 shows the sequences of the
oligonucleotides used for priming cDNA synthesis. T4 polynucleotide
kinase and [32P]ATP were used for 5'-end labeling of
oligonucleotides; this was followed by electrophoresis on 20%
polyacrylamide gels or affinity chromatography (30). For
primer extension, 8.9 × 104 cpm of labeled primer was
annealed to 5 µg of total RNA at 65°C for 10 min and cDNA was
synthesized at 48°C for 60 min with Moloney murine leukemia virus
reverse transcriptase (Superscript H
; Gibco-BRL,
Gaithersburg, Md.). The extended products along with the DNA sequence
(Sequenase; U.S. Biochemicals, Cleveland, Ohio) of the SalI
C fragment of VZV DNA were resolved on sequencing gels (11).
Northern blot analysis.
Total RNA (20 µg) from
VZV-infected and control BSC-1 cells was resolved by electrophoresis in
1% agarose gels containing 0.5 mM methylmercury(II) hydroxide (Johnson
Matthey, Ward Hill, Mass.), transferred to Zeta-Probe membranes
(Bio-Rad, Hercules, Calif.), and probed with either the entire VZV gene
21 ORF, which extends from nucleotide (nt) 30759 to nt 33872 on the VZV
genome (14), or a human 
-actin cDNA (10, 12).
Double-stranded DNA probes were radiolabeled with
[32P]dCTP by nick translation (30).
Plasmid construction.
The initiation codons for VZV ORFs 20 and 21 are located at nt 30475 and nt 30759, respectively, and the ORFs
are oriented in opposite directions (14). The 284-bp
intergenic region separating ORFs 20 and 21 was amplified from pBSalC
(11) by PCR with oligonucleotide primers containing either
KpnI or MluI restriction endonuclease sites
(Table 1). PCR conditions were reported previously (12). The
PCR product was resolved by agarose gel electrophoresis, digested with
KpnI and MluI, and inserted into a promoterless
chloramphenicol acetyltransferase (CAT) reporter plasmid (pCAT3basic;
Promega, Madison, Wis.). Before the PCR product was inserted into the
reporter plasmid, the multiple cloning site was modified to invert the orientation of the KpnI and MluI sites. Inversion
of the KpnI and MluI sites of pCAT3basic was
obtained by digesting pCAT3basic with KpnI and
MluI and then ligating a double-stranded adapter consisting
of annealed oligonucleotides KpnI-MluI-p1 and
KpnI-MluI-p2 (Table 1). The fidelity of PCR and
cloning was verified by DNA sequencing.
To construct plasmids placing VZV ORFs 4, 10, 61, 62, and 63 under the
control of the cytomegalovirus (CMV) IE promoter (pCIneo; Promega), the
VZV ORFs, along with 6 to 1,817 bp of flanking sequences, were shuttled
into the vector, pAlter-1 (Promega). Following DNA sequencing to
determine the orientation of the insert, single-stranded phage DNA and
oligonucleotide primers (Table 1) were used to synthesize chimeric
double-stranded plasmid DNA. After transformation of Escherichia
coli INV
F' (In Vitrogen, San Diego, Calif.) and selection of
tetracycline-sensitive, ampicillin-resistant organisms (antibiotic
switch), the plasmid inserts were partially sequenced to confirm the
introduction of the desired restriction endonuclease site. The VZV ORFs
were then shuttled from pAlter-1 to pCIneo and again the inserts were
partially sequenced to confirm the correct construct. Oligonucleotide
primers used to introduce the restriction endonuclease sites were
designed to leave unchanged the virus DNA sequence around the
initiation codon.
DNA transfection and reporter gene assay.
BSC-1 cells
(0.7 × 106 cells) were seeded into 90-mm-diameter
dishes and grown for 18 to 24 h at 37°C in a humidified
CO2 incubator. Supercoiled plasmid DNA, extracted by
affinity chromatography (Qiagen, Santa Clarita, Calif.), was diluted to
20 µg in 500 µl of Hanks balanced salt solution. Each transfection
reaction mixture consisted of 15 µg of reporter plasmid and 5 µg of
-galactosidase-expressing plasmid DNA (pSV-Gal; Promega) and was
precipitated at room temperature for 20 min with the addition of
CaCl2 to 0.124 M (20). The DNA-CaPO4 precipitate was added directly to the cell monolayer, and cells were
harvested and lysed 48 h after transfection and assayed for total
protein and -galactosidase activity (30). Cell extracts were diluted to yield equal amounts of -galactosidase activity, and
acetylation of [14C]chloramphenicol was determined by
ascending thin-layer chromatography (30). Where promoter
activity was determined as a response to transactivation by various VZV
proteins, equal amounts of cell protein extract were used in CAT
assays.
| |
RESULTS |
VZV gene 21 transcriptional unit.
Northern blot analysis of
total RNA extracted from VZV-infected cells demonstrated that the VZV
gene 21 transcript is a single species of approximately 3.1 kb (Fig.
1). We have previously determined the
3'-terminal structure of VZV gene 21 from both lytically infected cells
in tissue culture and from latently infected human trigeminal ganglia
(9, 12). The VZV gene 21 ORF is followed by 45 to 52 nt of
untranslated RNA containing a typical eukaryotic polyadenylation signal
and a poly(A)+ tail. To determine the structure of the VZV
gene 21 transcript at the 5' end, primer extensions were used in which
RNA extracted from productively infected cells was reverse transcribed
with various 32P-end-labeled oligonucleotides complementary
to ORF 21 (Table 1). The sizes of the extended products were compared
to those of a DNA sequencing ladder obtained by sequencing the
SalI C fragment of VZV DNA with each primer. Figure
2 shows that for each primer used, the
reverse transcription product terminated at the identical adenosine (nt
30681), located at 79 with respect to the initiation codon for VZV
gene 21. Faint bands were observed at 110 with respect to the
initiation codon for VZV gene 21 when the VZV-infected BSC-1 RNA was
extended with primer 21pe1 and at 117 when the RNA was extended with
primer 21pe2. These products may indicate the presence of minor gene 21 transcripts initiating from a subordinate TATA box; however, since they
are not coterminal and not observed when primer 21pe3 is used to extend
VZV-infected BSC-1 RNA, these products may be artifacts of the reverse
transcription reaction. No product was observed when the primers were
used to extend uninfected BSC-1 cell RNA. Identical results were
obtained when the experiment was repeated with a different preparation
of infected cell RNA. Thus, the VZV gene 21 transcript is a single
3.1-kb poly(A)+ RNA containing a 3,113-nt ORF bounded by
untranslated regions of 79 nt at the 5' end and 45 to 52 nt at the 3'
end.
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FIG. 1.
Northern blot analysis of VZV-infected BSC-1 RNA. (A)
Total RNA (20 µg) extracted from control BSC-1 cells (lanes C) and
VZV-infected BSC-1 cells (lanes V) along with RNA standards (lane M;
Gibco-BRL) was resolved in 1% agarose gels containing 0.5 mM
methylmercury(II) hydroxide and stained with 0.5 µg of ethidium
bromide per ml in 0.5 M ammonium acetate. (B and C) RNA was transferred
to a nylon-based membrane and probed for VZV gene 21 transcripts (B) or
-actin transcripts (C). VZV gene 21 transcripts are visible as a
discrete 3.1-kb band in VZV-infected cell RNA. Both control and
VZV-infected cell RNA contain discrete 1.8-kb -actin transcripts.
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FIG. 2.
Location of the 5' start of RNA transcription for VZV
gene 21. Total RNA (5 µg) from either VZV-infected (lanes V) or
uninfected (lanes C) BSC-1 cells was annealed to oligonucleotide primer
21pel, 21pe2, or 21pe3 end labeled with 32P (Table 1).
First-strand cDNA was synthesized, and the extended product was
resolved by gel electrophoresis. The DNA sequence of the
SalI C fragment of VZV DNA primed with the respective
oligonucleotides was used to size the cDNA products. The entire gel
image as well as an enlargement of the region containing extended
products is shown. With all three primers, the cDNA product obtained
from VZV-infected cell RNA (closed arrows in lanes V) terminated at the
identical adenosine located at nt 30681 on the VZV genome. Minor
extended products obtained from VZV-infected cell RNA (open arrows in
lanes V) were also observed. No product was observed when uninfected
cell RNA was used in the cDNA synthesis reaction (lanes C).
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The VZV gene 21 promoter is silent in uninfected cells.
To
investigate the VZV gene 21 promoter, the 284-bp DNA segment separating
ORF 20 and ORF 21 (Fig. 3) was cloned
into the CAT reporter plasmid. Figure 4
shows that in control BSC-1 cells, the VZV gene 21 promoter induces CAT
at levels beneath detection under the circumstances used. Further, the
function of a VZV-induced protein is required for gene 21 promoter
activity.
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FIG. 3.
Schematic representation of the VZV DNA between ORFs 20 and 21. The VZV genome consists of unique long (UL) and
unique short (US) segments of DNA, each bounded by inverted
and repeated DNA sequences (TRL/IRL and
IRS/TRS). ORFs 20 and 21 are oriented in
opposite directions, and both map within the SalI C fragment
(positions 23454 to 35936) within the UL. The 284-bp DNA
segment separating ORFs 20 and 21 contains one potential IE62 binding
site and three TATAA-like boxes. The 5' start site of gene 21 transcription is located at nt 30681, and the 3' end of the transcript
has been mapped to nt 33888 and nt 33895 (7, 10). The
boundary of the CAT reporter constructs used to locate the VZV gene 21 promoter are shown.
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FIG. 4.
The VZV gene 21 promoter is silent in uninfected cells.
The 284-bp VZV DNA segment separating ORFs 20 and 21 was inserted into
the CAT reporter plasmid and used to transfect either uninfected cells
(lane 2) or VZV-infected cells (lane 4). Controls included the CAT
reporter plasmid lacking a promoter transfected into uninfected cells
(lane 1) or VZV-infected cells (lane 3) and a CMV IE promoter driving
CAT transfected into uninfected cells (lane 5). CAT assays were
performed in duplicate, and the average acetylation of chloramphenicol
(%CAT) showed that the VZV gene 21 promoter does not function in
uninfected cells (VZV) but is active in VZV-infected cells (+VZV).
The amount of promoter activity in infected cells above that in
uninfected cells (fold) showed that gene 21 promoter activity was
approximately 650-fold higher in VZV-infected cells than in uninfected
cells.
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The VZV gene 21 promoter is transactivated by the protein encoded
by ORF 62 (IE62).
Since the VZV gene 21 promoter is silent in
uninfected cells and active during virus infection, one or more
VZV-induced proteins must function to transactivate the gene 21 promoter. The most likely candidates are VZV IE proteins or the
tegument-associated transactivating protein (encoded by ORF 10).
Therefore, we constructed expression plasmids in which VZV IE protein
genes corresponding to ORF 4, 61, 62, and 63, along with ORF 10, were
placed under the control of the CMV IE3 promoter. Western blot analysis
was used to confirm the ability of each construct to express the
respective protein in transient-transfection assays (data not shown).
Figure 5 shows that the VZV gene 21 promoter is silent in uninfected BSC-1 cells or in BSC-1 cells
expressing ORF 4, 10, 61, or 63. However, cotransfection of BSC-1 cells
with the gene 21 promoter-CAT construct with the ORF 62 expression
plasmid transactivated the VZV gene 21 promoter 42-fold more than
cotransfection with the gene 21 promoter alone.
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FIG. 5.
The VZV gene 21 promoter is transactivated by IE62. CAT
reporter plasmids, either promoterless (lane 1) or containing the
284-bp VZV ORF 20-ORF 21 intergenic region (lanes 2 to 7) or the CMV IE
promoter (lane 8), were transfected into cells either alone (lanes 1, 2, and 8) or with plasmids expressing various VZV transactivators (ORFs
4, 10, 61, 62, and 63) (lanes 3 to 7). Duplicate CAT assays indicated
that the VZV gene 21 promoter is transactivated by VZV IE62 but not by
the proteins encoded by VZV genes 4, 10, 61, and 63. Transactivation of
VZV gene 21 promoter by IE62 is ~42-fold higher than VZV gene 21 promoter activity in the absence of IE62. %CAT, average percent
chloramphenicol acetylation; sd, standard deviation.
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5' boundary of the gene 21 promoter.
Figure
6 shows the results of transient
transfection of BSC-1 cells with various 5' truncations of the VZV ORF
20-ORF 21 intergenic region inserted into the CAT reporter
plasmid. Inspection of the ORF 20-ORF 21 intergenic region indicates
the presence of three TATAA-like boxes. The plasmids were constructed
to eliminate successively these putative transcriptional regulatory
elements. To determine if transcriptional regulatory elements exist
upstream of the ORF 20-ORF 21 intergenic region that controls
expression of gene 21, a further CAT construct that extended 496 bp
into the 5' end of ORF 20 was made (p21Z-CAT). Since we had determined
that IE62 is required for gene 21 promoter activity, all transfections
included the IE62 expression vector. The CAT activity of p21Z-CAT was
similar to that of p21-CAT, indicating that the VZV gene 21 promoter is contained entirely within the segment of DNA spanning ORFs 20 and 21. Deletion of the 111 bp from position 30475 to position 30585 had little
effect on VZV gene 21 promoter activity and reduced CAT activity only
from 90.6 to 80.8%. Deletion of the 152 bp from position 30475 to
position 30626, however, had a marked effect on VZV gene 21 promoter
function and reduced its activity to background levels. Further 5'
truncations of the VZV gene 21 promoter also resulted in background CAT
activities. These results indicate that the 5' boundary of the gene 21 promoter regulatory region of the VZV gene 21 promoter lies between nt
30585 and nt 30626. Since this region contains a TATAA box that could
direct the 5' start of transcription, plasmid p21
, which consists of
the DNA segment from nt 30475 to nt 30597 inserted into the CAT
reporter plasmid, was constructed. In transient-cotransfection assays, p21 demonstrated no CAT gene translation products in the presence of
IE62 (Fig. 6), indicating a lack of promoter activity.
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FIG. 6.
5' boundary of the gene 21 promoter. Duplicate CAT
assays were performed on extracts of uninfected cells that had been
transfected with CAT reporter constructs containing either the entire
284-bp DNA segment separating ORF 20 and ORF 21 (p21), a 496-bp
extension (p21Z), or various 5' truncation mutations (p21A, p21B, p21C,
and p21) in the presence of the IE62 expression plasmid. The 5'
boundary of the gene 21 promoter was located to a region between nt
30585 (p21A) and nt 30626 (p21B). %CAT, average percent
chloramphenicol acetylation; sd, standard deviation.
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| |
DISCUSSION |
VZV gene 21 consists of a 3,113-nt ORF bounded by a 5'
untranslated region of 79 nt and by a 3' untranslated region of 45 to
52 nt. During productive infection in tissue culture, VZV gene 21 transcripts appear as a single, discrete 3.1-kb band on denaturing agarose gels. We produced antibodies in rabbits directed against VZV
gene 21-glutathione S-transferase fusion proteins and
located gene 21 protein predominantly in the cytoplasm as well as in
the nucleus of productively infected cells (29a). Although
the protein encoded by VZV gene 21 has not been studied, it is
homologous (47%) to HSV-1 UL37 (40), a 120-kDa
phosphoprotein synthesized after the onset of virus DNA replication
(1). In HSV-1-infected cells, UL37 is both cytoplasmic and
nuclear and incorporates into the tegument of progeny virions (31,
38). HSV-1 UL37 and ICP8 (the VZV gene 29 product homolog) form a
DNA-binding complex (39, 40).
During VZV latency, polyadenylated transcripts mapping to VZV gene 21 are present in human trigeminal ganglia (9, 12). While no
animal model of VZV latency and reactivation currently exists, simian
varicella virus (SVV) infection in monkeys closely mimics VZV infection
in humans (18), and polyadenylated transcripts corresponding
to the SVV homolog of VZV gene 21 have been demonstrated in monkey
ganglia latently infected with SVV (7).
The consistent detection of VZV gene 21 transcripts in latently
infected human ganglia (9, 10, 12) and its SVV homolog in
latently infected monkey ganglia (7) suggests that VZV gene 21 is vital to the maintenance of varicella latency or that its transcription is constitutive because cellular transcription factors recognize the promoter. We have identified the VZV gene 21 promoter and
have shown that it is silent in uninfected cells, indicating that its
activity depends upon a virus-induced protein. We have further
identified IE62 as the virus protein capable of transactivating the VZV
gene 21 promoter. Along with VZV gene 21 transcripts, polyadenylated
transcripts mapping to ORF 29 have been detected in latently infected
human ganglia (9, 32). Like the promoter for VZV gene 21, the VZV gene 29 promoter is silent in uninfected cells but is
transactivated by VZV IE62 (33, 34). VZV IE62 is a
promiscuous transactivator that recognizes numerous VZV, HSV-1, human
immunodeficiency virus, and cellular promoters (22, 23, 36).
VZV IE62 DNA binding has been located to a nonpalindromic pentamer,
ATCGT, and inspection of the VZV gene 21 promoter region shows this
potential binding site for region II of IE62 (4, 41, 42).
However, deletion of the potential IE62 binding site did not diminish
the transactivation of gene 21 promoter by IE62. VZV IE62
transactivation has also been associated with the ubiquitously present
cellular transcription factor USF (34). However, the minimal
VZV gene 21 promoter domain responsive to IE62 transactivation lacks
the consensus USF DNA binding sequence CACGTG. Thus, the promoter for
gene 21 may unlock a novel mechanism by which IE62 maintains gene
regulation.
This work was supported in part by Public Health Service grants
AG 06127 and NS 32623 from the National Institutes of Health.
We thank Paul Kinchington for antisera against proteins encoded by VZV
ORFs 4, 10, 61, and 62. We also thank Mary Devlin for editorial review
and Cathy Allen for preparation of the manuscript.
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Abstract
Introduction
