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J Virol, March 1998, p. 1910-1917, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Temporal Gene Regulation of the Channel Catfish
Virus (Ictalurid Herpesvirus 1)
Suming
Huang
and
Larry A.
Hanson*
College of Veterinary Medicine, Mississippi
State University, Mississippi State, Mississippi 39762
Received 9 June 1997/Accepted 24 November 1997
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ABSTRACT |
To identify promoter regions that impart differential temporal
regulation of channel catfish virus (CCV) genes, the transcriptional kinetics of an immediate-early gene and prospective early and late
genes were characterized. A cDNA clone, designated IE3C, representing a third immediate-early transcript was identified. The 5'
end of the IE3C transcript was mapped to nucleotides 15,368 and 131,043 in the terminal repeat regions of the CCV genome. The full length of
the transcript represented by the IE3C clone is 1,412 bp, and it most
likely codes for the protein specified by open reading frame (ORF) 12. The putative product of ORF12 contains a consensus RING finger metal
binding motif (C3HC4 structure). Temporal
expression studies, in conjunction with protein synthesis and DNA
replication inhibition, demonstrated that the IE3C transcript belongs
to an immediate-early kinetic class, the ORF5 transcript is a member of
the early kinetic class, and ORF39 and ORF46 are true
late-kinetic-class genes. Additionally, we demonstrated that ORF38
transcription overlaps ORF39 and the products presumably share the same
poly(A) signal. The 5' ends of the transcripts encoding ORF38, ORF39,
and ORF46 were mapped to nucleotides 44,862, 45,254, and 59,644, respectively, and potential transcriptional control elements
were located.
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INTRODUCTION |
Channel catfish virus (CCV), or
ictalurid herpesvirus 1, is a cytopathic herpesvirus that causes a
severe hemorrhagic disease in young channel catfish, Ictalurus
punctatus (58). CCV is the most intensely studied
herpesvirus of lower vertebrates. The entire genome has been sequenced,
and it has been predicted to contain 79 genes, 14 of which are located
in the terminal-repeat regions (10). The genomic structure
of CCV is much different from those of identified herpesviruses of
mammalian or avian species (10).
Herpesvirus gene expression is coordinately regulated and sequentially
ordered such that the genes can be classified into three broad temporal
groups, immediate-early (IE), early, and late genes. IE gene expression
initiates the viral lytic cascade (28, 29). The induction of
early and late viral genes depends on viral regulatory proteins encoded
by IE genes that act in trans. The IE genes are expressed
first and are defined as capable of being transcribed in the absence of
de novo viral protein synthesis (28, 29).
The initiation of early and late-gene expression, unlike that of the IE
genes, is not homogeneously defined. The early gene products function
in nucleotide metabolism and viral DNA synthesis, and some downregulate
the expression of IE genes (7, 37, 38, 42, 59). The late
genes, expressed last and requiring prior early gene expression,
generally encode the virion structural proteins. They either may be
expressed in the absence of viral DNA synthesis (delayed early class)
or may stringently require viral DNA synthesis (true late class)
(31, 32, 36).
Kinetic analysis of the synthesis of CCV polypeptides revealed three
distinct groups of proteins differing in their time of appearance and
magnitude of synthesis. These results suggested that CCV protein
synthesis is coordinately regulated (13). Recently, two IE
transcripts were characterized and reported as representing open
reading frame 8a (ORF8a) and ORF9 of the terminal-repeat regions of the
CCV genome (53). However, the structure, function, and
temporal expression class of most CCV-encoded proteins have yet to be
determined. Because CCV is evolutionarily distant from mammalian
herpesviruses, its conserved regulatory mechanism might provide insight
into the factors influencing herpesvirus evolution. Exploring the
differential temporal gene regulation of CCV is the first step in
dissecting the virus-associated regulatory mechanisms controlling CCV
infection. In this study, representative transcripts of the IE, early,
and late genes of CCV were identified, their temporal regulation was
characterized, and promoter regions were predicted from the CCV genome
sequence. The results confirm that CCV gene expression is sequentially
ordered and regulated in a manner similar to that of mammalian
herpesviruses. We also identified a third IE gene.
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MATERIALS AND METHODS |
Cells and viruses.
The CCV strain used in this experiment
was Auburn clone A (American Type Culture Collection). Channel catfish
ovary (CCO) cells and their thymidine kinase (TK)-negative counterpart
(CCOBr cells) were cultured as described previously (23).
When a specific chemical inhibitor of transcription or DNA replication
was used, the chemical was added to the cell culture medium and the
cells were incubated with the chemical for 1 h before infection,
as well as during infection and for a specified time postinfection.
The replication of CCV DNA in CCOBr cells over time or in the presence
of acyclovir (ACV) (Sigma Chemical Co., St. Louis, Mo.) or
phosphonoacetic acid (PAA) (Sigma Chemical Co.) was analyzed by the
trichloroacetic acid (TCA) precipitation assay as described previously
(49) and by slot blot DNA-DNA hybridization. For the TCA
precipitation assay, the cells were cultured in medium containing 10 µCi of [methyl-3H]thymidine per ml (48 Ci/mmol) and infected with 10 PFU of CCV per cell. Three replicate cell
samples were harvested and lysed at serial time points. Viral DNA was
precipitated with 10% TCA on G6 glass fiber filters (Fisher
Scientific, Pittsburgh, Pa.). The slot blot assays were performed by
infecting monolayered CCO cells in 24-well plates with 3 PFU of CCV per
cell in 150 µl of medium per well for 30 min, aspirating off the
inoculum, and overlayering with 1 ml of fresh medium per well. At the
appropriate time, the medium was aspirated off, the cells were lysed,
and DNA was purified by using a Puregene kit (Gentra Systems Inc.,
Minneapolis, Minn.). One-half of the DNA purified from each cell
culture well was transferred to a positively charged nylon membrane
(Zeta Probe GT; Bio-Rad Laboratories, Hercules, Calif.) slot in a slot
blot (1). CCV DNA was detected by using a nonradioisotopic
DNA-DNA hybridization kit (ECL Direct Nucleic Acid Labelling Systems;
Amersham International, Buckinghamshire, England) with
EcoRI-digested cosmid pHCCV 386 (22) as a probe
and Hyperfilm-ECL (Amersham International).
Isolation of RNA.
The CCO cells were infected with 10 PFU of
CCV per cell in the presence or absence of 100 µg of cycloheximide
per ml for 8 h. The total RNA was isolated by the guanidinium
thiocyanate method (8). The mRNA was purified from total RNA
by using a PolyATtract mRNA isolation system (Promega, Madison, Wis.).
Construction of an IE-enriched cDNA library.
A monolayer of
CCO cells was treated with 50 µg of cycloheximide (Sigma Chemical
Co.) per ml, infected with 10 PFU of CCV per cell, and incubated at
30°C for 6 h. The cells were then lysed, and mRNA was obtained
by guanidinium thiocyanate lysis and oligo(dT) hybridization-mediated
magnetic separation (PolyATtract System 1000; Promega). The cDNA was
produced from poly(A) RNA by using Moloney murine leukemia virus
reverse transcriptase (Promega) and oligo(dT) primer. The cDNA library
was cloned into the specialized bacteriophage 
vector Zap II,
using a kit ( Zap Gold) as described by the manufacturer
(Stratagene, La Jolla, Calif.). CCV-specific cDNA clones were
identified by using nonradioisotopically labeled purified CCV DNA in
plaque hybridization analyses with the ECL kit (Amersham Corporation,
Arlington Heights, Ill.). Positive plaques were excised and
bacteriophage was eluted. Then pBluescript SK portions of these clones
were rescued by using ExAssist helper phage in the SOLR strain of
Escherichia coli (Stratagene).
Nested DNA deletion and sequencing.
Nested deletion
subclones of the IE3C cDNA were generated by using an exonuclease
III-mung bean nuclease deletion kit (Stratagene) according to the
manufacturer's instructions after digestion with SacI and
EcoRI, generating a unique 3'-overhang restriction site and
a unique 5' restriction site between the insert and the 3' site chosen.
These fragments were sequenced by using a Sequenase version 2.0 DNA
sequencing kit (United States Biochemical, Cleveland, Ohio) with 1,000 Ci of [
-35S]dATP (Amersham Co.) per mmol and T3 or T7
primers according to manufacturer's instructions. The sequence data
were connected using Contig Manager of the DNASIS version 3.0 program
(Hitachi Software Engineering America, Ltd., San Bruno, Calif.).
Identification of 5' ends of transcripts.
The 5' ends of IE
and late transcripts were obtained by the 5' rapid amplification of
cDNA ends (RACE) method, using the 5'-AmpliFINDER RACE kit (Clontech
Laboratories, Inc., Palo Alto, Calif.). For evaluation of the
full-length IE3C transcript, first-strand cDNA was synthesized from 6 µg of total RNA isolated from cycloheximide-restricted CCV-infected
CCO cells, using Moloney murine leukemia virus reverse transcriptase
(Promega) and the P1 primer (for the primer sequence, see
Table 1). The amplification step used the anchor primer (Clontech) and
the P2 primer (Fig. 1B).
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FIG. 1.
Diagram of the CCV genome demonstrating the locations
and orientations of ORF5, ORF12, ORF39, ORF38-ORF39, and ORF46 and the
primers and riboprobes used in this study. (A) The CCV genome with the
unique region (solid line) and direct-repeat regions (open boxes), and
expanded depictions of the regions covered by ORF5, ORF12, ORF38-ORF39,
and ORF46, with arrows indicating the direction of transcription. (B)
The primers used for cloning and 5' RACE. (C) Riboprobes used for RNase
protection assays and Northern blot analysis. The restriction sites
used in the cloning procedures are indicated.
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The 5' transcription start sites of ORF39 and ORF46 were mapped
similarly, using an avian myeloblastosis virus reverse transcriptase
(Clontech)-generated cDNA template produced from 1 µg of mRNA
from
CCV-infected CCO cells. P
4 and P
5 were used for
ORF39 and
ORF46 cDNA synthesis, respectively. The custom primers
P
4 and
P
5 were used in the amplification step
for ORF39 and ORF46, respectively
(Fig.
1B).
Construction of plasmids.
Plasmid pBSCV552, containing the
EcoRI-to-XhoI fragment of the IE3C cDNA, plasmid
pBSCV553, containing the BamHI-to-PstI fragment of the CCV TK gene, and plasmid pBSCV543, containing the
EcoRI-to-SpeI fragment of ORF46, were produced by
cloning the respective fragments into the multiple cloning site of
plasmid pBluescript SK
(Stratagene) (Fig. 1C). Plasmid
pBSCV605 was constructed by insertion of the 363 bp of PCR-amplified
ORF39 fragment, using primers CCV45342(+) and CCV45723(
) (Table
1), into SpeI and
EcoRI sites of pBluescript SK.
RNase protection assays and Northern blot analysis.
The
RNase protection assays were performed on lysates of CCOBr cell
monolayers grown in 25-cm2 flasks, exposed to 10 PFU of CCV
per cell, and harvested at serial time points, using a Lysate
Ribonuclease Protection Assay Kit in accordance with the
manufacturer's directions (Ambion, Austin, Tex.) and 0.23 µCi of
probe. After hybridization at 37°C overnight and RNase treatment, the
protected fragments were electrophoresed on a 5% polyacrylamide gel
containing 7% urea. The gel was autoradiographed with X-Omat film
(Kodak, Rochester, N.Y.).
The riboprobes antisense to ORF12 (IE3C), ORF5 (TK), ORF39, and ORF46
were derived from vectors described above. Riboprobes
were generated
and labeled with [
-
32P]UTP, using linearized plasmid
DNA, and transcribed with either
T3 or T7 RNA polymerase. Transcription
of pBSCV552, pBSCV553,
pBSCV605, and pBSCV543 generated riboprobes with
lengths of 495,
624, 426, and 229 nucleotides (nt) for ORF12, ORF5,
ORF39, and
ORF46, respectively (Fig.
1C).
Northern blot analysis was performed as previously described
(
49). Following the electrophoresis, the mRNA was
transferred
to Zeta-probe GT blotting membranes (Bio-Rad Laboratories).
The
ORF39 and IE3C riboprobes described above were used. Hybridization
was done at 70°C overnight in hybridization solution (0.25 M
Na
2HPO
4 [pH 7.2], 7% sodium dodecyl sulfate
[SDS]). The membrane was washed
twice in 20 mM
Na
2HPO
4 (pH 7.2) containing 5% SDS and then
twice
in 20 mM Na
2HPO
4 (pH 7.2) containing 1%
SDS at 65°C. The sizes
of Northern blot-detected transcripts were
determined by comparing
them to the RNA markers (Promega) that were run
on the same gel.
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RESULTS |
Identification of a CCV IE gene.
To identify and clone CCV IE
genes, a cDNA library was constructed from cycloheximide-restricted
CCV-infected CCO cells. The cDNA library consisted of 4.2 × 105 PFU of recombinant bacteriophage , which was
amplified to 3 × 108 PFU. Aliquots of 5.0 × 104 PFU were screened for clones containing CCV sequences
by plaque hybridization with purified CCV DNA as a probe. The internal
pBluescript plasmid of each of three isolated clones was rescued.
Sequencing and comparison to the CCV genomic sequence localized the
5'-3' coding strand sequence represented by one clone, IE3C, to regions 15,701 to 16,778 and 131,376 to 132,453 of the CCV genome. These regions span a portion of ORF12 through ORF13 of terminal-repeat portions of the CCV genome (10) (Fig. 1A).
Characterization of differential temporal transcripts.
To
evaluate differential gene regulation, the IE3C clone was chosen as a
representative IE transcript. The TK gene, ORF5 (10, 22),
was chosen as a potential early gene. A putative glycoprotein gene,
ORF46 (10), and ORF39, the major capsid protein gene
(11), were chosen as potential late genes. The transcription
of representative genes with predicted IE, early, and late regulation
was characterized by RNase protection assays and Northern blot
analysis. The assays were performed in conjunction with
[3H]thymidine incorporation and slot blot DNA-DNA
hybridization analysis of CCV DNA replication as a temporal reference
to distinguish early and late expression.
To characterize the production of the IE3C transcript, sequential
extracts of CCV-infected cells obtained with or without
cycloheximide
were analyzed by RNase protection assays (Fig.
2).
The IE3C riboprobe was protected by
transcripts from lysates obtained
as early as 0.5 h postinfection
(p.i.) (Fig.
2B, lane 0.5 h).
The concentration of transcripts
from this region peaked at 2.0
h p.i. and remained persistently
high through 8 h p.i. (Fig.
2A,
lanes 1 h through 2 h).
The IE3C transcripts were produced at
high levels with cycloheximide
inhibition (Fig.
2A, lane CHX).
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FIG. 2.
RNase protection and Northern blot analyses indicating
the kinetics of IE3C transcription. (A) Autoradiogram from an RNase
protection assay of cell lysates collected from uninfected cells
(Neg.), from cycloheximide treated infected cells (CHX), and from
infected cells at 0.5, 1, 2, 3, 4, 6, and 8 h p.i. (lanes 0.5 h through 8 h). Bands represent the 453-nt antisense
IE3C-protected RNA fragment (2-day film exposure) (see Fig. 1). Lane M,
molecular size markers; lane 3C, undigested probe. (B) IE3C detection
at 0.5 and 1 h p.i. (7-day film exposure). (C) Northern blotting
of poly(A) RNA from CCV-infected cells harvested at 3 and 8 h
p.i., using the IE3C antisense riboprobe. The 1,400-nt band
corresponding to the IE3C transcript is indicated by an arrow. The
predominant banding at 8 h p.i. ranges from 2,000 to 3,500 nt.
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Because RNase protection assays cannot differentiate overlying
transcripts, a Northern blot was performed on mRNA from CCV-infected
cell lysates at 3 and 8 h p.i., using the IE3C riboprobe indicated
in Fig.
1C. High-level expression of a 1,400-nt transcript predominated
at 3 h p.i., and the expression of larger transcripts predominated
by 8 h p.i. (Fig.
2C, lanes 3 h and 8 h, respectively).
The results
of the RNase protection assays indicate that the high level
of
transcription in the IE3C region at 8 h p.i. was apparently due
to overlapping transcripts. These results demonstrate
differential
transcription of the CCV IE3C gene region as the
infection progresses.
Initial IE promoter expression is apparently
reduced and production
of early and/or late transcripts overlapping the
IE3C ORF occurs
during later phases of the infection.
To determine the kinetic class of ORF5, ORF39, and ORF46
transcripts, RNase protection assays were performed on CCV-infected
cell lysates harvested from 0.5 to 16 h p.i. The ORF5 (TK)
transcript
was first detected at 1 h p.i. The concentration of
transcripts
from this region was highest at 1 and 2 h p.i. and
then decreased
(Fig.
3A, lanes 1h, 2h,
and 4h through 16h). Transcription of
ORF5 was inhibited by
cycloheximide (Fig.
3A, lane X). ORF39 and
ORF46 transcripts were first
detected at 3 h p.i., and the level
of these transcripts continued
to accumulate through 8 h p.i.
(Fig.
4A, lanes 3h through 8h). The time of
viral DNA replication
was determined by analyzing [
3H]dT
incorporation in CCV-infected CCOBr cells, using lysates
collected from
0.5 to 16 h p.i. Viral DNA replication was first
detected at
2 h p.i. (230 ± 12.7 cpm) and peaked at 6 h p.i.
(10,500
± 120 cpm) compared to the background (89 ± 8.8 cpm). The TCA
precipitation assay results were confirmed by DNA-DNA
hybridization
on slot blots of DNA purified from CCV-infected CCO cells
harvested
at 0, 1, 2, 3, 4, and 8 h p.i. The first detected signal
above
the 0-h-p.i. sample was at 3 h p.i.
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FIG. 3.
RNase protection assays indicating the kinetics of TK
gene transcription and the effect of cycloheximide and ACV on
transcription. (A) RNase protection of ORF5 antisense riboprobe (see
Fig. 1) when hybridized to infected-cell lysates harvested at various
times from 1 to 16 h p.i. (lanes 1h to 16h) or when cultured in
the presence of 100 µg of cycloheximide per ml for 8 h (X). (B)
Cell lysate RNase protection of the same riboprobe in CCV-infected cell
cultures incubated with incremental concentrations of ACV from 0 to 100 µM (lanes 0 through 100). Lane M, molecular size markers; lane P,
undigested probe.
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FIG. 4.
RNase protection assays indicating the kinetics of
synthesis of the ORF39 and ORF46 transcripts. Cell lysates were
collected from cycloheximide- or ACV-treated infected cells and from
infected cells at various times p.i. (A) Riboprobes designed to detect
ORF39 (363-nt protected fragment) and ORF46 (168-nt protected fragment)
were hybridized with RNA in lysates of cells treated with 100 µM
cycloheximide (X) and infected cells harvested at various times from 1 to 8 h p.i. (lanes 1h to 8h). (B) Riboprobes designed to detect
ORF39 and ORF46 (see Fig. 1) were hybridized with infected-cell
lysates, collected at 8 h p.i., from cultures incubated with ACV
at concentrations from 0 to 100 µM (lanes 0 through 100). P,
undigested probe; M, molecular size markers.
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To distinguish true late gene expression from delayed early expression,
viral DNA synthesis must be inhibited. Therefore,
the effect of ACV and
PAA on the synthesis of CCV DNA in CCOBr
cells was examined by
performing TCA precipitation assays. The
amount of
3H-labeled DNA from 10 µM ACV-treated infected cells was
reduced
almost 90% at 12 h p.i. (mean ± standard deviation,
7,730 ± 746
cpm in the 10 µM ACV-treated infected cells versus
66,600 ± 6,210
cpm in untreated infected cells). The amount of
3H-labeled DNA was reduced only about 45% in the presence
of 300
µg of PAA per ml (22,100 ± 6,100 cpm versus 48,900 ± 2,440 cpm
in untreated infected cells at 8 h p.i.). The results
indicate
that 10 µM ACV inhibited CCV DNA synthesis. DNA-DNA
hybridization
on slot blots of DNA purified from CCV-infected CCO cells
in 0,
5, and 10 µM ACV harvested at 8 h p.i. demonstrated
low-level
(in 5 µM ACV) or no (in 10 µM ACV) detectable CCV DNA
replication
compared to 0-h-p.i. controls.
When 10 µM ACV was used, production of the putative late transcripts
in CCV-infected cells was inhibited (Fig.
4B, lane 10)
while ORF5
transcripts continued to be produced (Fig.
3B, lane
10). Expression of
the ORF5 transcript required de novo viral
protein synthesis and was
independent of CCV DNA replication.
Therefore, the TK gene represented
an early class of CCV gene.
TK RNA synthesis was probably inhibited at
100 µM ACV because
of detrimental effects on the host cell at these
high concentrations.
In comparison, the expression of ORF39 and ORF46
transcripts exhibited
a stringent requirement for DNA replication,
defining these genes
as members of the true late class of CCV genes.
5'-End mapping of IE3C, ORF39, and ORF46 transcripts.
The 5'
terminus of the IE3C transcript was determined by the 5' RACE method,
using antisense nested primers P1 and P2 (Fig. 1B). The 3' nucleotide of P2 corresponded to nt 529 of the
IE3C cDNA. A single 854-bp PCR fragment which contained 327 bp upstream of the IE3C cDNA was amplified (Fig. 5).
This placed the transcriptional start site of IE3C at positions 15,368 and 131,043 of the CCV genome within the terminal-repeat ends. The
results of IE3C cDNA sequencing indicated that the full-length
transcript is 1,412 nt long. It is unspliced and contains ORF12 and
ORF13 (10). The putative start codon of ORF12 was located at
nt +35 of the transcript. Sequence analysis of the region upstream of
this transcriptional start site revealed one TATA-like sequence at nt
32, one core consensus sequence of enhancer at nt 321, and two Sp1
binding sites around the promoter (Fig.
6). The predicted protein encoded by the
IE3C gene is 299 amino acids long and contains a RING finger (C3HC4) metal binding motif near the amino
terminus (4, 20) (Fig. 6).
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FIG. 5.
A 1.2% agarose electrophoretic gel of the product of 5'
RACE of IE3C transcripts, using anchor and P2 primers
(left), and a sequencing gel of the cloned RACE product (right). The
arrow and asterisk indicate the transcriptional start site. 1Kb, 1-kb
DNA ladder (molecular size marker); 3C, 5' RACE product of IE3C
transcripts.
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FIG. 6.
Sequence analysis of the IE3C transcript and its
upstream promoter element. The TATA box, CCAT box, Sp1 element,
putative HSV enhancer core (enh.), and poly(A) signals (sig.) are
indicated. The RING finger (C3HC4) metal
binding motif is indicated by the boxes (4, 20). The stop
codon is indicated by an asterisk. The 5' limit of the cDNA clone IE3C
is indicated by a vertical line at nt 936. The arrow indicates the
transcription start site.
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The 5' end of the ORF46 transcript, mapped with nested primers
P
5 and P
6 (Fig.
1B), generated a single 611-bp
PCR fragment
(Fig.
7). The 5' end of the
ORF46 transcript corresponded to nt
59,644 of the CCV genome, which is
139 nt upstream of the putative
ORF46 start codon.
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FIG. 7.
A 1.2% agarose electrophoretic gel of the product of 5'
RACE of ORF39 and ORF46 (left) and sequencing gels of the cloned RACE
products, ORF38, ORF39, and ORF46 (from left to right). Arrows on gels
and asterisks in sequences indicate the transcriptional start sites.
The arrow on the ORF38 sequence indicates an extra guanosine. 1Kb, 1-kb
DNA ladder (molecular size marker).
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The 5' end of the ORF39 transcript, mapped with nested primers
P
3 and P
4 (Fig.
1B), generated 530- and 923-bp
fragments (Fig.
7). Sequence analysis placed the 5' end of the 530-bp
fragment
at nt 45,254 of the CCV genome, which is 63 bp upstream of the
putative ORF39 start codon. The 5' end of the 923-bp fragment
was
identified at nt 44,862 of the CCV genome, which is 40 nt
upstream of
the putative ORF38 start codon. The extra guanosine
between the 5' RACE
anchor sequence and the CCV sequence (Fig.
7) is assumed to represent
the methylated guanosine cap of the
mRNA. The extra guanosine has been
demonstrated in 5' RACE on
capped mRNA (
2). Northern blot
analysis of this region, using
the ORF39 riboprobe on mRNA from
CCV-infected cell lysates at
3 and 8 h p.i., demonstrated
low-level expression of a slightly
longer transcript at 3 h p.i.
compared with that at 8 h p.i. (Fig.
8). This supported the results of the 5'
RACE indicating that
the transcription of ORF38 overlaps ORF39.
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FIG. 8.
Northern blotting of poly(A) RNA from CCV-infected cells
harvested at 3 and 8 h p.i., using the ORF39 riboprobe. (Left
panel) Autoradiograph demonstrating expression levels at 3 and 8 h
p.i. X-ray film was exposed for 2 h. (Right panel) Use of
different film exposure times to demonstrate the size shift of the
riboprobe-specific mRNA. The 3-h-p.i. portion was autoradiographed for
14 h, and the 8-h-p.i. portion was autoradiographed for 20 min.
The positions of molecular size markers are shown on the left.
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DISCUSSION |
In this study, we demonstrated the presence of a third CCV-encoded
IE transcript. The total transcript sequence is 1,412 bp long. The 5'
end of the transcript is located at nt 15,368 and 131,043 in the CCV
genome. Two ORFs were predicted to lie in this region: ORF12 and ORF13
(10). The identified transcript likely encodes the ORF12
polypeptide. It is interesting that ORF12 and ORF13 are in the same
frame but read-through translation is not likely due to the presence of
three stop codons between the two ORFs (Fig. 6). As reported previously
(10), both putative polypeptides contain a potential zinc
metal binding motif near the amino terminus. The zinc binding motifs
were shown to be involved in protein-protein interactions and in
binding DNA and RNA (5). Two herpes simplex virus type 1 (HSV-1) IE proteins, ICP0 and ICP27, contain zinc metal binding domains
(17, 54, 55). The metal binding domain of the ORF12 product
is in the C3HC4 RING finger form similar to
ICP0 in HSV-1 and its homologs in other alphaherpesviruses (17,
20, 57). The presence of a putative RING finger metal binding
motif in the protein product and the high level of transcription without de novo protein synthesis (cycloheximide inhibition) suggest that ORF12 belongs to the IE family of CCV genes that may be involved in regulating the expression of other virus products.
Many IE proteins of herpesviruses have been shown to be important for
the transactivation of viral early genes and the progression of the
lytic replication cycle (16, 38, 47). HSV-1 encodes five IE
genes: 0, 4, 22, 27, and 47. The 4 gene encodes a
major regulatory protein, ICP4, which binds to viral DNA and regulates
viral genes both positively and negatively (12, 14, 18, 33, 35,
39-41). The 0 gene encodes a promiscuous transactivator (15, 43, 46). The 22 gene product is associated with
viral replication and optimal expression of 0 and late genes
(6). The 27 gene encodes a protein which regulates the
processing of viral RNA (50). Only the 47 gene encodes a
protein which does not have a known regulatory function. It blocks the
presentation of viral peptides to major histocompatibility complex
class I-restricted CD8+ T lymphocytes by associating with
peptide transporters (TAP) in the endoplasmic reticulum (21, 25,
60). It is also well understood that ICP4 is required for the
induction of TK expression (12, 44, 45). The HSV TK promoter
is often used to evaluate the regulation of eukaryotic gene expression
as well as its trans induction by viral regulatory proteins
(9). We have attempted to evaluate the effect of the product
of ORF12 on the expression of a lacZ reporter gene under the
control of the CCV TK promoter, using the plasmid pBSCV464
(61) and a construct containing ORF12 in the plasmid pBK-CMV
in cotransfection, transient expression assays; however, the assays
were hampered by the poor transfection efficiency in CCO cells.
Cotransfection, transient expression assays were also performed with
the COS cell line. The results indicated a decrease in lacZ
expression in the presence of the ORF12 product, but because of the
unnatural mammalian cell system used, the results were equivocal.
Considerable additional research on this gene and its potential
regulatory protein product is needed to determine their roles in the
progression of CCV gene expression.
Identification of the transcriptional start sites for ORF12, ORF38-39,
ORF39, and ORF46 has allowed comparison of upstream promoter
sequences of IE and late genes, as well as of well-characterized HSV IE- and late-promoter regulatory regions. The putative IE3C promoter includes a possible TATA box (CATAAA), a
likely CCAAT box (CGAAT), and two Sp1 elements surrounding the TATA
box. A core consensus transcriptional enhancer sequence,
5'-GTGGAAAG-3', was found within the nt 321 to 315
region of the IE3C mRNA (Fig. 9). The
core sequence is within the enhancer domains of a number of viruses,
including HSV IE gene promoter regions (34, 56). The AT-rich
homologs and GC-rich enhancer-like elements present in the IE3C
promoter region may be critical for transcription of the IE3C ()
gene.
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|
FIG. 9.
Comparison of CCV IE3C, ORF46, ORF38, and ORF39 promoter
regions. Arrows indicate the positions of transcription start sites.
The scale, in nt upstream of the mRNA start sites, is indicated at the
top. The TATA box, CCAAT box, Sp1 element, and putative HSV enhancer
core (Enh.) are indicated.
|
|
Inspection of the region upstream of the ORF46 transcriptional start
site revealed that an Sp1 element and a CCAAT box were located upstream
of a TATA-like sequence (TATTAA) (Fig. 9). It is unusual for
a true late gene to contain two recognized upstream transcription-regulatory sequences. The well-characterized true late
HSV-1 promoters contain only one TATA-like sequence with no other
recognizable upstream cis-acting regulatory elements (3, 19, 26, 27, 31, 52). The structure of the ORF46 promoter
is similar to that of the well-characterized HSV early promoters of
UL23 (TK), UL9, UL8, and UL29 (ICP8) (3).
The ORF38 promoter region is similar to ORF46 in that it has a
CCAAT-like sequence upstream of the respective TATA-like
sequence, TATTAA (Fig. 9). The structure of the ORF38
promoter is closer to that of herpesvirus early promoters, which
contain cis-acting regulatory elements. The early-promoter
characteristic of ORF38 is supported by Northern blot analysis data,
which showed low-level expression of ORF38 during early gene
expression.
The putative ORF39 promoter includes a TATA-like sequence, TAATTT,
with no other recognizable upstream cis-acting
regulatory elements (Fig. 9), similar to HSV true late-promoter
sequences (3, 19, 26, 27, 31, 52). ORF39 encodes the major capsid protein of CCV (11). In this study, we also
demonstrated that the expression of the ORF39 transcript stringently
requires viral DNA synthesis, suggesting that ORF39 belongs to the true late family of CCV genes.
CCV DNA sequence analysis by Davison (10) indicated that
ORF38 and ORF39 share one poly(A) signal, AATAAA, located at
nt 48,759 (10). Our results support this. Two transcripts
were detected by 5' RACE using ORF39 primers P3 and
P4 (Fig. 1B), and both were detected by Northern blot
analysis. Overlapping transcription of the ORF38-ORF39 gene region is
likely, due to the absence of poly(A) signals between ORF38 and ORF39.
Temporal transcriptional evaluation and the associated effects of viral
protein synthesis and DNA replication inhibition demonstrate that CCV
transcriptional regulation is similar to that of mammalian herpesviruses. The identification of a third IE gene indicates that CCV
may have a complex, interactive form of IE-mediated gene regulation
similar to that of HSV-1 (47), bovine herpesvirus 1 (51), equine herpesvirus 1 (24), and
varicella-zoster virus (48) but in contrast to that of
pseudorabies virus, which has only one IE gene (30).
| |
ACKNOWLEDGMENTS |
This research was primarily supported by the National Research
Initiative Competitive Grant Program/USDA (94-37204-0853). Support was
also provided by Mississippi Agricultural and Forestry Experiment
Station (MAFES) project MISV-0892 and the College of Veterinary
Medicine, Mississippi State University.
We thank Mary Rudis and Suzana Marinovic for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: College of
Veterinary Medicine, Mississippi State University, P.O. Box 9825, Mississippi State, MS 39762. Phone: (601) 325-1202. Fax: (601)
325-1031. E-mail: hanson{at}cvm.msstate.edu.
Mississippi Agricultural and Forestry Experiment Station
publication J-9180.
Present address: Department of Medicine/Hematology, Vanderbilt
University Medical Center, Nashville, TN 37232-2279.
| |
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Abstract
Introduction
