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Journal of Virology, April 2000, p. 3659-3667, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Rta of Murine Gammaherpesvirus 68 Reactivates the
Complete Lytic Cycle from Latency
Ting-Ting
Wu,1
Edward J.
Usherwood,2
James P.
Stewart,3
Anthony A.
Nash,3 and
Ren
Sun1,*
Department of Molecular and Medical
Pharmacology, the UCLA AIDS Institute, the Jonsson Comprehensive Cancer
Center, and the Molecular Biology Institute, University of California
at Los Angeles, Los Angeles, California 900951;
The Trudeau Institute, Saranac Lake, New York
129832; and Department of Veterinary
Pathology, University of Edinburgh, Edinburgh EH9 1QH, United
Kingdom3
Received 7 October 1999/Accepted 21 January 2000
 |
ABSTRACT |
Herpesviruses are characterized as having two distinct life cycle
phases: lytic replication and latency. The mechanisms of latency
establishment and maintenance, as well as the switch from latency to
lytic replication, are poorly understood. Human gammaherpesviruses, including Epstein-Barr virus (EBV) and human herpesvirus-8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), are
associated with lymphoproliferative diseases and several human tumors.
Unfortunately, the lack of cell lines to support efficient de novo
productive infection and restricted host ranges of EBV and HHV-8 make
it difficult to explore certain important biological questions. Murine
gammaherpesvirus 68 (MHV-68, or 
HV68) can establish de novo lytic
infection in a variety of cell lines and is also able to infect
laboratory mice, offering an ideal model with which to study various
aspects of gammaherpesvirus infection. Here we describe in vitro
studies of the mechanisms of the switch from latency to lytic
replication of MHV-68. An MHV-68 gene, rta (replication and
transcription activator), encoded primarily by open reading frame 50 (ORF50), is homologous to the rta genes of other
gammaherpesviruses, including HHV-8 and EBV. HHV-8 and EBV Rta have
been shown to play central roles in viral reactivation from latency. We
first studied the kinetics of MHV-68 rta gene transcription
during de novo lytic infection. MHV-68 rta was
predominantly expressed as a 2-kb immediate-early transcript. Sequence
analysis of MHV-68 rta cDNA revealed that an 866-nucleotide
intron 5' of ORF50 was removed to create the Rta ORF of 583 amino
acids. To test the functions of MHV-68 Rta in reactivation, a plasmid
expressing Rta was transfected into a latently infected cell line,
S11E, which was established from a B-cell lymphoma in an
MHV-68-infected mouse. Rta induced expression of viral early and late
genes, lytic replication of viral DNA, and production of infectious
viral particles. We conclude that Rta alone is able to disrupt latency,
activate viral lytic replication, and drive the lytic cycle to
completion. This study indicates that MHV-68 provides a valuable model
for investigating regulation of the balance between latency and
lytic replication in vitro and in vivo.
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INTRODUCTION |
Latency provides unique advantages
for herpesviruses to escape host immune surveillance and to
establish lifelong persistent infections. However, to maintain viral
reservoirs and transmit to other hosts, herpesviruses must be
reactivated from latency and enter the lytic replication phase to
generate more virus. The balance between viral latency and lytic
replication is therefore a critical factor that determines the outcome
of infection and the corresponding pathogenesis. If the balance favors
lytic replication, lytic infections of herpes simplex virus sometimes
lead to morbidity through encephalitis or visual loss through
keratoconjunctivitis (43). On the other hand, if the balance
favors viral latency, latent infection by Epstein-Barr virus (EBV) can
cause lymphoproliferative diseases (29).
The physiological signals that cause reactivation of herpesviruses are
not well understood. The molecular mechanisms of reactivation have been
most extensively investigated in two human gammaherpesviruses, EBV and
human herpesvirus 8 (HHV-8). Most of these studies have been carried
out in B-cell lymphoma-derived cell lines harboring the latent virus.
In EBV, two viral gene products, ZEBRA and Rta, are expressed earliest
upon reactivation induced by chemical or biological agents
(21, 25, 33, 37) and activate viral promoters triggering
lytic gene expression (1-3, 5, 6, 11, 12, 14). To study the
functions of ZEBRA and Rta in reactivation, plasmids expressing ZEBRA
and Rta were transfected into latently infected B-cell lines to
determine whether expression of viral lytic genes and lytic replication
of viral DNA were activated. ZEBRA alone is able to activate viral
lytic cycle in B cells and epithelial cells latently infected with EBV
(5, 15, 19). Rta synergizes with ZEBRA to promote activation
of viral lytic gene expression (3, 6, 27, 44), but does not
always disrupt latency by itself. In certain latently infected B-cell and epithelial cell lines, Rta can disrupt viral latency (28, 44). ZEBRA and Rta act as transcriptional activators in transient transfection assays with reporter constructs. Moreover, ZEBRA and Rta
have been shown to stimulate expression, not only of themselves, but of
each other (8, 28, 32, 44, 45), although the levels of
activation vary, depending upon the experimental system used.
Therefore, it has been proposed that ZEBRA and Rta function in a
cooperative manner to activate the viral lytic cycle.
The HHV-8 homologue of EBV Rta has been shown to be sufficient to
activate expression of early and late viral lytic genes in B-cell lines
latently infected with HHV-8; however, it has not been demonstrated
whether viral lytic DNA replication or virus production can be induced
(17, 35). Upon reactivation, HHV-8 rta is
expressed as an immediate-early gene, but the zebra
homologue of HHV-8 is an early gene. Moreover, HHV-8 ZEBRA is not able
to disrupt latency (35). Although the roles of ZEBRA and Rta
in reactivation of EBV or HHV-8 have been investigated, their
expression and functions during de novo lytic infection cannot be
studied, because there is no efficient in vitro system available. In
addition, the lack of an effective animal model has made studies of
gammaherpesvirus reactivation in vivo almost impossible.
Murine gammaherpesvirus 68 (MHV-68, also referred to as HV68), which
is phylogenetically related to HHV-8 and EBV, offers an excellent model
in which to study the mechanisms underlying the dynamic balance between
latency and lytic replication (40). In vitro cell culture
systems are available to study de novo lytic infection, latency, and
reactivation of MHV-68. Moreover, MHV-68 can establish lytic and latent
infection in laboratory mice (36), which allows us to
address questions regarding the host-virus interactions (22, 23,
30, 33). MHV-68 forms plaques on monolayers of many cell lines,
making it relatively easy to genetically manipulate the viral genome.
This also makes it possible to examine the functions of individual
viral genes in various aspects of the viral life cycle, including reactivation.
The molecular mechanisms of MHV-68 reactivation, however, have
not been previously characterized. Efforts have been made to identify
the zebra homologue in MHV-68, but so far have not been successful. On the other hand, the rta homologue is readily
found in MHV-68 (18), and, in fact, rta is
conserved among gammaherpesviruses, including EBV, HHV-8,
herpesvirus saimiri (HVS), and bovine herpesvirus 4 (BHV-4). This led
us to test the hypothesis that MHV-68 Rta may be the central viral
factor governing reactivation. In this study, the kinetics of
MHV-68 rta transcription during de novo lytic
infection were first examined. Next, the functions of MHV-68 Rta in
reactivation were studied by using a B-cell line harboring latent
MHV-68. Our results indicate that MHV-68 rta is expressed as
an immediate-early gene during de novo lytic infection and is capable
of initiating viral lytic replication in latently infected B cells.
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MATERIALS AND METHODS |
Viruses, cells, and plaque assays.
MHV-68 was
originally obtained from the American Type Culture Collection (VR1465),
and the working stocks were grown by infecting BHK-21 cells (ATCC
CCL-10) at 0.1 PFU per cell. BHK-21 cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal
bovine serum (FBS). S11E is a clonal cell line of S11, which was
established from a B-cell lymphoma developed in an MHV-68-infected mouse and contains latent MHV-68 (38). S11E was cloned on
the basis of a low spontaneous reactivation frequency (13).
S11E cells were cultured in RPMI 1640 medium containing 15% FBS. To infect BHK-21 cells, the viral inoculum in DMEM was incubated with
cells for 1 h with occasional swirling. The inoculum was removed
and replaced with fresh DMEM plus 10% FBS. For the experiments involving cycloheximide (Sigma, St. Louis, Mo.) (see Fig. 2), cells
were treated at a concentration of 100 or 200 µg/ml 1 h prior
to, during, and after viral inoculation until they were harvested. For
the experiments using phosphonoacetic acid (PAA) (Sigma) (Fig. 2),
cells were treated at a concentration of 200 µg/ml after viral
inoculation until they were harvested.
The viral titers were measured by plaque assays, using monolayers of
BHK-21 cells overlaid with 1% methylcellulose (Sigma) for 5 days. The
cells were fixed and stained with 2% crystal violet in 20% ethanol.
Plaques were then counted to determine the titers. To measure the viral
titers in the supernatants of transfected S11E cells, the suspension
cultures of cells were centrifuged twice at 450 × g, and
the supernatants were harvested for plaque assays.
RT-PCR, DNA cloning, and sequencing.
For reverse
transcription (RT), 0.5 µg of 22-mer oligo(dT) and 2 µg of total
RNA isolated from BHK-21 cells infected with MHV-68 (2 PFU/cell) at
8 h postinfection were first denatured at 70°C for 10 min,
immediately placed on ice, and incubated with 200 U of Superscript II
(Gibco BRL, Gaithersburg, Md.) at 42°C for 1 h. Ten percent of
the product was then amplified by PCR, with primers R3
(5'-CTGAATTCGCAGCGATGGCCTCTGACTC-3',
containing an EcoRI site [underlined] and the
Kozak's sequence upstream of the translation initiation codon
[boldface], corresponding to nucleotide [nt] 66760) and R4
(5'-GATCTAGACCGTTTATGACTCCAGGCTG-3', containing an XbaI site [underlined] upstream of the
termination codon [boldface], corresponding to nt 69374). The RT-PCR
product was reamplified by using R3 and another primer internal to R4, R6 (5'-GATATGTACCCACATGGATGCCTGT-3', corresponding to nt
67974 to 67950). The resulting product was cloned into the pCR2.1
T-vector (Invitrogen, Carlsbad, Calif.). The positive colonies were
selected by blue-white screening, and plasmid DNA from those colonies
was isolated for DNA sequencing with primer R7
(5'-CATCTTCAGGGTGCTGTAGGAA-3', corresponding to nt
67781 to 67760). The nucleotide numbering is according to Virgin et al.
(40), and the relative positions of the primers to open
reading frame 50 (ORF50) are indicated in Fig. 1C. To construct an Rta
expression vector, the rta genomic sequence was amplified by
PCR with primers R3 and R4 from total DNA isolated from MHV-68-infected
BHK-21 cells. The 2.6-kb PCR product was subjected to XbaI
digestion followed by EcoRI digestion and then cloned into a
pcDNA3 vector (Invitrogen) containing the cytomegalovirus
immediate-early promoter and enhancer.
RNA extraction and Northern analysis.
Total RNA was
extracted from BHK-21 or S11E cells by the guanidinium-acid phenol
method, as described by Chomczynski and Sacchi (4). RNA was
treated with a mixture of 1 M glyoxal and 50% (vol/vol) dimethyl
sulfoxide at 50°C for 30 min (7). Glyoxalated RNA was then
separated on 1% agarose gels in circulating 10 mM sodium phosphate
buffer (pH 6.8). A 1-kb ladder (Gibco BRL) and 
HindIII were 5' end labeled with
[-32P]dATP, glyoxalated, and loaded onto the gels as
the size standards. RNAs on gels were transferred to charged nylon
membranes (Amersham Pharmacia Biotech, Arlington Heights, Ill.). The
membranes were UV cross-linked and deglyoxalated at 80°C in 20 mM
Tris-HCl (pH 8). Prehybridization and hybridization were carried out at
65°C in 0.5 M K2HPO4 (pH 6.8) containing 7%
sodium dodecyl sulfate (SDS) and 1% bovine serum albumin. The probes
were generated by the random-priming method with
[
-32P]dCTP with the templates generated by PCR of
viral genomic DNA. The membranes were then washed at 65°C with 40 mM
sodium phosphate (pH 6.8) containing 5% SDS and 0.5% bovine serum
albumin, followed by washing with 40 mM sodium phosphate (pH 6.8)
containing 0.5% SDS. Radioactivity was detected and quantitated with a
STORM imaging system (Molecular Dynamics, Sunnyvale, Calif.). Before
rehybridization with a different probe, the membranes were stripped at
80°C in 10 mM Tris-HCl (pH 8) containing 1% SDS.
Transfection.
Transfection of S11E cells was carried out by
electroporation. S11E cells (107) and 10 µg of plasmid
DNA were mixed in a cuvette (Bio-Rad, Hercules, Calif.) and shocked in
a Gene-Pulser II (Bio-Rad). To monitor transfection efficiencies, 2 µg of plasmid pEGFP-C1 (Clontech, Palo Alto, Calif.) was included in
each transfection, and the percentage of cells expressing green
fluorescent protein was determined at 24 h posttransfection by
fluorescent microscopy.
Western analysis.
Cells were lysed in Laemmli buffer
containing 0.25 M Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5%
-mercaptoethanol, and 0.002% bromophenol blue. The lysates were
heated to 95°C and subjected to electrophoresis on 10%
polyacrylamide gels. The broad-range prestained protein standard
(Bio-Rad) was also loaded onto the gels. Proteins on gels were
electrotransferred (Bio-Rad) onto nitrocellulose membranes (Amersham
Pharmacia Biotech). The membranes were blocked in phosphate-buffered
saline (PBS) plus 0.1% Tween 20 and 5% milk, incubated with the
rabbit hyperimmune serum against MHV-68-infected rabbit cells
(36), washed in PBS containing 0.1% Tween 20, and incubated
with antirabbit immunoglobulin G conjugated with horseradish
peroxidase. The proteins were detected with the enhanced
chemiluminescence detection ECL+PLUS system (Amersham Pharmacia
Biotech), and the signals were imaged with a STORM imaging system
(Molecular Dynamics).
DNA extraction and Southern analysis.
Total DNA was
harvested by lysing cells in the buffer containing 10 mM Tris-HCl (pH
8), 50 mM EDTA (pH 8), and 0.5% SDS. The lysates were incubated with
proteinase K (100 µg/ml) at 50°C overnight and then extracted twice
with phenol-chloroform (1:1). DNA was precipitated with ammonium
acetate and ethanol, and the DNA pellet was dissolved in Tris-EDTA
buffer (pH 8). Total DNA was subjected to restriction enzyme digestion
overnight and electrophoresed on 0.8% agarose gels. Gels were stained
with ethidium bromide to visualize DNA and then subjected to
depurination, denaturation, and neutralization. DNAs on treated gels
were transferred to charged nylon membranes (Amersham Pharmacia
Biotech). The membranes were UV cross-linked and prehybridized at
68°C in the buffer containing 5× SSC (1× SSC is 0.15 M NaCl plus
0.015 M sodium citrate), 10× Denhardt's solution, 0.5% SDS, and
denatured salmon sperm DNA (50 µg/ml). Probes were generated by the
random priming method using [-32P]dCTP and PCR
products of genomic viral DNA as templates. The membranes were washed
at 68°C in 2× SSC with 0.1% SDS, followed by 0.1× SSC with 0.1%
SDS. Radioactivity was detected and quantitated by using a STORM
imaging system (Molecular Dynamics).
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RESULTS |
The structure of the rta gene.
MHV-68 ORF50
was previously shown to share homology to ORF50 of HHV-8, EBV, and HVS
(18, 40). The genomic location of ORF50 is shown in Fig.
1A. ORF50 encodes the major portion of the rta homologue of gammaherpesviruses. The rta
genes of HHV-8, EBV, HVS, and BHV-4 share one common feature: the
second exon containing ORF50 is spliced to the first exon by removing
an intron carrying ORF49 (17, 19, 35, 39, 41). Because of
the conservation of the splicing event, we hypothesized that the
rta gene of MHV-68 undergoes similar RNA processing. To
confirm this, total RNA was isolated from MHV-68-infected BHK-21 cells
and reverse transcribed with oligo(dT), followed by PCR, with primers
flanking ORF50 (R3 and R4). The resultant RT-PCR product was ~1.7 kb
and smaller than the PCR product (2.6 kb) generated with the same pair
of primers, but with genomic DNA as the template, consistent with RNA
splicing (data not shown). To precisely determine the splicing
junction, the RT-PCR product was reamplified with primer R3 and another
primer, R6 (internal to R4), and then cloned and sequenced with the R7
primer. As shown in Fig. 1B, the splicing donor and acceptor sites are
located at nt 66795 and 67661, respectively (numbering according to the
National Center for Biotechnology Information database
[40]). The sequences flanking nt 66795 and 67661 were
highly homologous to the consensus sequences flanking the splice donor
and acceptor sites, respectively. As a result of splicing, an 866-nt
intron containing the entire ORF49 was removed (Fig. 1C). Instead of
the predicted methionine of ORF50 (40), the translation
initiation methionine of MHV-68 rta would be located in the
5' small exon at nt 66760. Consequently, MHV-68 Rta would have an extra
94 amino acids (aa) added to the N terminus of ORF50 (489 aa).
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FIG. 1.
Structure of the MHV-68 rta gene. (A) Genomic
location of ORF50. The nucleotide numbers are assigned on the basis of
the MHV-68 sequence in the National Center for Biotechnology
Information database (40). The open boxes represent ORFs
predicted by computer analysis, and the numbers assigned to individual
ORFs are based on homology with the corresponding ORFs of HVS and HHV-8
(40). The arrows in the open boxes indicate the orientation
of ORFs. A major portion of the rta gene is carried by
ORF50. Downstream of ORF50 is ORFM7 encoding gp150, which shares
homology with EBV gp350 (34). (B) Sequencing analysis of the
rta cDNA. RNA harvested from BHK-21 cells infected with
MHV-68 at 2 PFU/cell was reverse transcribed with oligo(dT) and
amplified, first by primers R3 and R4 and then by primers R3 and R6.
The resultant product was cloned, and the positive clone was sequenced
with the R7 primer. The splice donor and acceptor sites were mapped to
nt 66795 and 67661, respectively. (C) Splicing of the rta
gene. The splice donor (s.d.) and splice acceptor (s.a.) sites were
localized to nt 66795 and 67661, respectively. An intron of 866 nt is
removed, and two exons are joined together to generate the Rta ORF,
with the translation initiation codon at nt 66760. Primers R3 and R4
were used for PCR amplification of the cDNA product and the genomic
sequence spanning the rta gene. R3 and R6 were used to
reamplify the cDNA PCR product of R3 and R4 for cloning. R7 was used to
sequence the splicing junction. The probe shown in panel C was used for
Northern analysis of the rta transcripts shown in Fig. 2A.
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MHV-68 rta was expressed as an immediate-early
transcript.
To examine the kinetics of MHV-68 rta
transcription during de novo infection, BHK-21 cells were infected at 5 PFU/cell, and total RNA was harvested at different times postinfection.
In addition, infections were carried out in the presence of 100 or 200 µg of cycloheximide per ml (a protein synthesis inhibitor) or 200 µg of PAA per ml (a herpesvirus DNA polymerase inhibitor). The
rta transcripts were analyzed by Northern blotting with the
0.7-kb PCR fragment spanning the C terminus of ORF50 (corresponding to nt 68651 to 69378; Fig. 1C) as a probe. As shown in Fig.
2A, a major band of 2.0 kb was detected
at the earliest time point, 2 h postinfection (Fig. 2A, lane 4).
Expression of the 2-kb transcript peaked at 4 h postinfection
(Fig. 2A, lane 5) and decreased at 8 h postinfection (Fig. 2A,
lane 6). In the presence of cycloheximide, the 2-kb transcript was
still detected (Fig. 2A, lanes 7 and 8), consistent with the expression
pattern of an immediate-early gene.
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FIG. 2.
MHV-68 rta is expressed as an immediate-early
gene. Infection was carried out in six-well plates, and RNA was
harvested at various times postinfection (p.i.), as indicated at the
top of the panels. Half of the total cellular RNA was glyoxalated and
subjected to Northern analysis. In each panel, lanes 1 and 2 are a 1-kb
DNA ladder and HindIII, respectively; the sizes of
the individual bands are indicated to the left. RNA was collected at 0 (lane 3), 2 (lane 4), 4 (lane 5), or 13 (lane 9) h postinfection. RNA
was harvested from cells at 8 h postinfection without (lane 6 [N]) or with cycloheximide treatment at 100 (lane 7 [C1]) or 200 (lane 8 [C2]) µg/ml. At 24 h postinfection, RNA was isolated
from cells without (lane 10 [N]) or with (lane 11) 200 µg of PAA
per ml added to the medium. The same membrane was hybridized with three
different probes, as shown in the three panels. The probe used in panel
A was derived from the very last 0.7-kb sequence (nt 68651 to 69378) or
ORF50, which was generated by SacI digestion of the PCR
product amplified with the R3 and R4 primers. Panel B shows the image
of the membrane rehybridized with the probe derived from the PCR
product spanning the 1.9-kb TK ORF (nt 32879 to 34813). The probe used
in panel C was derived from the PCR product of the 0.5-kb M9 ORF (nt
93962 to 94522). The deduced sizes of the transcripts are indicated to
the right of each panel. Radioactivity was detected with a STORM
imaging system.
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A single-stranded RNA probe was used for Northern analysis (data not
shown) and confirmed that the orientation of the 2-kb
transcript was
the same as that of ORF50. This 2.0-kb transcript
would be large enough
to encode the entire ORF of Rta (583 aa).
The 0.7-kb probe also
detected a few minor larger transcripts,
one of which was deduced to be
3.4 kb and only appeared in the
cells treated with cycloheximide (Fig.
2A, lanes 7 and 8). Other
transcripts appeared later than the major
2-kb transcript, and
their expression was abolished in the presence of
cycloheximide
(Fig.
2A, lanes 7 and 8), as well as in the presence of
PAA (Fig.
2A, lane 11), indicating that they were late viral
transcripts.
The same membrane was stripped and rehybridized with a probe derived
from the thymidine kinase (TK) ORF (Fig.
2B). Several
transcripts were
detected, with three predominant species of 2.1,
2.6, and 3.8 kb. The
major 2.6-kb species was detected at 2 h
postinfection (Fig.
2B,
lane 2) and was expressed earlier than
the other species of 2.1, 2.8, 3.2, and 3.8 kb, but disappeared
at 8 h postinfection (Fig.
2B,
lane 6). Its expression, however,
was sensitive to cycloheximide
treatment (Fig.
2B, lanes 7 and
8), indicating that this was an early
viral transcript. The minor
3.2-kb species was expressed at kinetics
similar to those of the
major 2.6-kb transcript and was also an early
viral transcript.
Other species of transcripts, including the other
major 2.1- and
3.8-kb transcripts, continued to accumulate until
13 h postinfection
and then decreased at 24 h postinfection.
However, their expression
was inhibited by the presence of PAA (Fig.
2B, lane 11), leading
to the conclusion that the other species were
late viral transcripts.
Our results are consistent with a previous
study in which several
TK-related transcripts were detected, including
a 2.6-kb transcript,
and the expression pattern of this 2.6-kb
transcript was characteristic
of an early gene (
26).
The membrane was rehybridized with a probe derived from the M9 ORF
(Fig.
2C). MHV-68 M9 has homology to ORF65 of HHV-8 (
40),
which encodes a capsid protein. Several species of RNAs were detected,
including two major transcripts of 0.9 and 3.6 kb, which were
observed
at 4 h postinfection, gradually increased until 13 h
postinfection and then declined by 24 h postinfection. Their
expression
was abrogated by treatment with PAA (Fig.
2C, lane 11),
indicating
that they were late viral transcripts. Two minor transcripts
of
2.8 and 2.9 kb were also observed. The 2.8-kb RNA was detected
at
4 h and disappeared at 24 h postinfection, and the 2.9-kb RNA
was not visible until 8 h, and continued to accumulate until
13
h postinfection. Expression of both RNAs was sensitive to
the
presence of PAA (Fig.
2C, lane 11), indicating that, like the
two
major transcripts, these two minor transcripts were late viral
RNAs.
MHV-68 Rta activated viral lytic gene expression in latently
infected S11E cells.
To study the function of MHV-68 Rta, the
2.6-kb sequence (nt 66760 to 69373) spanning the initiation methionine
and the termination codon was cloned into a eukaryotic gene expression
vector, pcDNA3, where expression was driven by the cytomegalovirus
immediate-early promoter. Since rta was expressed as
an immediate-early gene during de novo lytic infection, we tested
whether Rta could disrupt latency and initiate the cascade of lytic
gene expression. pcDNA3/MHV-68 rta was transfected
into an MHV-68 latently infected B-cell line, S11E, and transfection
efficiency was determined by the percentage of cells expressing green
fluorescent protein encoded by a cotransfected plasmid, ranging from 5 to 10%.
Transfected S11E cells were harvested at different times
posttransfection for Western analyses by using the rabbit hyperimmune
serum raised against MHV-68-infected rabbit cell lysates, and
the
results are shown in Fig.
3. Transfection
of pcDNA3/MHV-68
rta (Fig.
3A) into S11E cells induced the
expression of a variety
of proteins, which were also seen in lytically
infected BHK-21
cells (Fig.
3A, lane 2). Expression of most proteins
peaked at
24 h posttransfection, although some proteins were
visible as
early as 12 h posttransfection. No viral lytic proteins
were detected
in pcDNA3-transfected S11E cells at any time
posttransfection
(Fig.
3B). Because 10
5 S11E cells with a
10% transfection efficiency were loaded in
each lane (Fig.
3A, lanes 4 to 6), we found that much stronger
signals were detected in a similar
number of infected BHK-21 cells
(10
4) in the control
(Fig.
3A, lane 2), indicating higher levels of
lytic protein expression
in these cells. It was also noted that
one lytic protein (Fig.
3A,
indicated with an asterisk) was expressed
at high levels in infected
BHK-21 cells, but was not detected
in S11E cells transfected with
pcDNA3/MHV-68
rta.
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FIG. 3.
Induction of viral lytic proteins by Rta. S11E cells
(107) were electroporated with pcDNA3/MHV-68 rta
(A) or pcDNA3 (B). Total protein from 105 cells was
collected at 12 (lanes 4 of panels A and B), 24 (lanes 5 of panels A
and B), or 48 (lanes 6 of panels A and B) h posttransfection (p.t.).
Proteins from 104 uninfected (UI [lane 1]) or
MHV-68-infected (I [lane 2]) BHK-21 cells or from 105
untransfected S11E cells (UT [lanes 3]) were loaded and subjected to
Western analysis with the rabbit hyperimmune serum against
MHV-68-infected rabbit cell lysates. The molecular masses (kilodaltons)
of individual proteins in the marker are indicated to the left.
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Northern analyses were also performed to examine lytic gene
transcription in transfected S11E cells. The kinetics of TK and
M9 gene
expression after Rta transfection are shown in Fig.
4.
As described earlier, five major
TK-related transcripts were seen
in lytically infected BHK-21 cells
(Fig.
2B), and a similar pattern
of TK transcripts was also detected in
S11E cells transfected
with pcDNA3/MHV-68
rta (Fig.
4A,
lanes 6 to 9), but not in cells
transfected with pcDNA3 (Fig.
4A, lanes
2 to 5). The 2.6- and
3.2-kb transcripts expressed as early transcripts
in lytically
infected BHK-21 cells (Fig.
2B) were induced to a high
level as
early as 12 h posttransfection, and their expression
decreased
at 24 h posttransfection. The other TK-related RNAs,
expressed
as late viral transcripts in lytically infected BHK-21 cells
(Fig.
2B), were visible at 12 h posttransfection, but continued to
accumulate
until 24 h posttransfection.
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FIG. 4.
Activation of viral lytic gene expression by Rta. RNA
from S11E cells electroporated with pcDNA3 (lanes 2 to 5) or
pcDNA3/MHV-68 rta (lanes 6 to 9) was collected at 12 (lanes
2 and 6), 24 (lanes 3 and 7), 36 (lanes 4 and 8), or 48 (lanes 5 and 9)
h posttransfection (p.t.). Half of the total cellular RNA was
glyoxalated and subjected to Northern analysis. The same membrane
hybridized with different probes is shown in two panels. The probe used
in panel A is derived from the TK ORF (as in Fig. 1B). In panel B, the
probe is derived from the M9 ORF (as in Fig. 1C). Lane 1 is a 1-kb DNA
ladder, with the sizes of individual bands indicated to the left. The
deduced sizes of transcripts are indicated to the right. Radioactivity
was detected with a STORM imaging system.
|
|
The same membrane was stripped and rehybridized with the M9 ORF probe
(Fig.
4B). In lytically infected BHK-21 cells, the major
0.9- and
3.6-kb M9 transcripts were expressed as late viral RNAs
(Fig.
2C). As
seen in Fig.
4B, transfection of pcDNA3/MHV-68
rta (Fig.
4B,
lanes 6 to 9), but not of pcDNA3 (Fig.
4B, lanes 2 to
5), led to
expression of 0.9- and 3.6-kb M9 transcripts in S11E
cells. The
transcripts accumulated to high levels at 24 h posttransfection.
Two minor M9 RNAs seen in lytically infected BHK-21 cells were,
however, only detected at very low levels in S11E cells transfected
with pcDNA3/MHV-68
rta.
MHV-68 Rta induced viral DNA replication in latently infected S11E
cells.
Since transfection of pcDNA3/MHV-68 rta
activated some late viral transcripts, such as those of M9, the
expression of which was demonstrated earlier to be dependent on viral
DNA replication in lytically infected BHK-21 cells (Fig. 2C, lanes 10 and 11), viral DNA replication might also have been induced in S11E
cells transfected with pcDNA3/MHV-68 rta. To confirm this,
viral DNA replication was analyzed by Southern blotting. As seen in
Fig. 5, viral DNA in S11E cells was
greatly amplified by transfection of pcDNA3/MHV-68 rta
(lanes 6 to 8), but not by pcDNA3 (lanes 3 to 5). At 24 h
posttransfection, a 90-fold increase in the amount of viral DNA in
pcDNA3/MHV-68 rta-transfected cells was detected compared to
the amount in untransfected cells (Fig. 5, lanes 2 and 6). Given that
the transfection efficiency was 10%, this would be an average of
900-fold induction of viral DNA synthesis in each transfected cell.
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|
FIG. 5.
Amplification of viral DNA by Rta. Total DNA from 2 × 106 S11E cells transfected with pcDNA3 (lanes 3 to 5) or
pcDNA3/MHV-68 rta (lanes 6 to 8) was harvested at 24 (lanes
3 and 6), 48 (lanes 4 and 7), or 72 (lanes 5 and 8) h posttransfection
(p.t.) and subjected to digestion with HindIII. The
digested DNAs were electrophoresed on a 0.7% agarose gel for Southern
analysis. HindIII-digested DNA from 5 × 105 MHV-68-infected BHK-21 cells was loaded in lane 1 as a
positive control (I), and digested DNA from 2 × 106
untransfected S11E cells was loaded in lane 2 as a negative control
(UT). The probe used to detect the viral DNA was derived from the
6.2-kb PCR product (nt 51 to 6298) spanning the far left end of the
viral genome. Based on the sequence (40), a 6.2-kb DNA
fragment would be detected with the probe. Radioactivity was detected
and quantitated with a STORM imaging system.
|
|
To confirm that the increase in viral DNA by Rta transfection was due
to the induction of viral lytic DNA replication rather
than
amplification of the viral latent genome, a terminal repeat
assay
(
31) was performed. The linear MHV-68 genome has multiple
1.2-kb tandem repeats at each terminus. In latently infected S11E
cells, the MHV-68 genome exists as an episomal circular DNA generated
by fusion through the termini of the linear form (
38). As
illustrated
in Fig.
6A, the terminal
repeat assay is based on the fact that
herpesviral DNA is replicated
via a rolling-circle mechanism during
the lytic cycle. Monomers of
viral genomic DNA with variable numbers
of terminal repeats are
produced as a result of DNA cleavage at
any one of the terminal repeats
between monomeric viral genomes
on concatemers. After digestion of
viral DNA with
HindIII (not
present in the repeat) and
probing with a unique region adjacent
to terminal repeats, a 1.2-kb
ladder of DNA fragments would be
generated, due to variable numbers of
repeats at the terminus.
If lytic replication does not occur, after
digestion of circular
viral DNA, a single large DNA fragment containing
the fused termini
that harbors multiple copies of terminal repeats
would be detected
on Southern blots. The results of the terminal repeat
assay are
shown in Fig.
6B. Since viral DNA was greatly increased by
Rta
at 24 h posttransfection (Fig.
5), DNA was harvested at an
additional
time point at 12 h posttransfection. In S11E cells
transfected
with pcDNA3, only a large DNA fragment (
23 kb) was
detected at
all time points (Fig.
6B, lanes 2 to 5), indicating that
lytic
replication did not occur. However, the DNA of cells transfected
with pcDNA3/MHV-68
rta gave rise to ladders similar to those
seen
in lytically infected BHK-21 cells (Fig.
6B, lane 1). The
intensity
of DNA laddering was increased between 12 and 24 h
posttransfection
and then declined. These results provide evidence that
transfection
of Rta induced lytic replication of viral DNA and
processing of
the replicated viral DNA.
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|
FIG. 6.
Induction of lytic replication and processing of viral
DNA by Rta. (A) The basis of the terminal repeat assay. The solid lines
represent the double-stranded viral genome, and the boxes represent
terminal repeats. In panel A, a rolling-circle mechanism is illustrated
at the top, and the right ends of monomeric viral genomes after DNA
processing are shown at the bottom. Since DNA processing can occur at
any one of the terminal repeats between monomeric viral genomes on
concatemers, there will be various numbers of repeats, with as few as
one, at the termini of monomeric viral genomes. The gray arrow and
dotted line indicate where digestions of HindIII occur
adjacent to the terminal repeats. The shaded box indicates the position
of the probe used for panel B. The details are further described in the
text. (B) The results of the terminal repeat assay. Total DNA from
106 S11E cells transfected with pcDNA3 (lanes 2 to 5) or
pcDNA3/MHV-68 rta (lanes 6 to 9) was harvested at 12 (lanes
2 and 6), 24 (lanes 3 and 7), 36 (lanes 4 and 8), or 48 (lanes 5 and 9)
h posttransfection (p.t.). DNA was digested with HindIII
and subjected to Southern analysis with a probe (its relative position
is indicated in panel A) derived from a 0.8-kb PCR product (nt 118314 to 117560). Lane 1 is the positive control from the DNA of
105 MHV-68-infected BHK-21 cells digested with
HindIII (I). Indicated to the left are the sizes of
individual bands in a 1-kb DNA ladder. Radioactivity was detected with
a STORM imaging system.
|
|
MHV-68 Rta transfection led to production of infectious viral
particles from latently infected S11E cells.
We have shown that
introduction of MHV-68 Rta into S11E cells activated viral lytic gene
expression and DNA replication. We further tested whether Rta was
sufficient to drive the viral lytic cycle to production of viruses. The
supernatants from transfected S11E cells were collected at
various time points, and the viral titers were measured by plaque
assays. The results are shown in Fig. 7.
The viral titers in the supernatants from Rta-transfected S11E
cells were much higher than in those from pcDNA3-transfected S11E
cells. At 48 h posttransfection, there were ~130-fold more infectious viruses in the supernatant from Rta-transfected S11E cells.
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|
FIG. 7.
Production and release of infectious viruses induced by
Rta. At the times indicated at the bottom of the diagram, 10 ml of
suspension cultures of S11E cells transfected with pcDNA3 (squares) or
pcDNA/MHV-68 rta (diamonds) was centrifuged twice to remove
cells, and the supernatants were collected for plaque assays to measure
the viral titers. The data, including standard deviations indicated
with error bars, were obtained from two or three independent assays and
are expressed as PFU per milliliter of supernatant.
|
|
| |
DISCUSSION |
Previous studies have shown the central roles of HHV-8 or
EBV Rta in the switch from viral latency to lytic replication.
Since the rta gene is highly conserved among
gammaherpesviruses, we studied the gene expression and functions
of MHV-68 rta in cell cultures. MHV-68 rta
was expressed as an immediate-early gene during de novo lytic infection
of BHK-21 cells. Furthermore, exogenous expression of Rta activated the
virus from latency in S11E cells harboring latent MHV-68. Our results
have provided a foundation for studying the molecular mechanisms
controlling establishment, maintenance, and disruption of latency in vivo.
Our analysis of the MHV-68 rta gene demonstrates that an
866-nt intron containing the entire ORF49 is spliced, generating the
Rta ORF of 583 aa with the initiation methionine at nt 66760 (Fig. 1).
5' rapid amplification of cDNA ends was also carried out on the
polyadenylated RNA, and the preliminary data localized the 5' end of
the rta transcript at ~100 nt upstream of the initiation methionine of the Rta ORF (data not shown). Taken together with a
consensus poly(A) signal located 57 nt after the termination codon, the
size of the spliced rta RNA is estimated to be ~1.9 kb,
consistent with the size of the major immediate-early rta transcript (2 kb) detected in lytically infected BHK-21 cells (Fig.
2A). However, the nature of the other minor transcripts requires
further investigation.
The rta genes of gammaherpesviruses share similarities in
the genomic location, amino acid sequence, and splicing pattern, which
suggests that Rta plays a conserved and important role in the life
cycle of the virus. However, among gammaherpesviruses, there are
differences in terms of the location of the initiation methionine and
the nature of the rta transcript. Except for EBV (19), the initiation methionines of Rta in MHV-68, HHV-8
(17, 35), HVS (42), and BHV-4 (39) are
located in the first rather than the second exon. Thus, EBV Rta is
entirely encoded by ORF50 (19), whereas the Rta proteins of
the other gammaherpesviruses have extra amino acids added to the N
terminus of ORF50. The rta transcripts of MHV-68, HVS, and
BHV-4 are monocistronic (39, 41), whereas the EBV
rta transcript is bicistronic, with an additional ORF
encoding ZEBRA downstream of the rta ORF
(19). The HHV-8 rta transcript is
polycistronic, with not only the K8 ORF encoding the ZEBRA
homologue (10, 16, 17, 35, 46), but also the putative
K8.2 ORF downstream of the K8 ORF (46). It is not clear
whether ZEBRA of EBV or HHV-8 can be translated from either
the bicistronic or polycistronic message. No ZEBRA homologue has been
identified in MHV-68, HVS, or BHV-4.
MHV-68 Rta most likely functions as a transcriptional activator.
Homologues of Rta from other gammaherpesviruses such as EBV, HVS, and
BHV-4 have been shown to activate the promoters of viral early genes in
transient transfection assays (1, 9, 11, 12, 24, 39). Amino
acid sequence alignments of the Rta homologues reveal that the most
conserved region is at the N terminus. This conserved portion of EBV
Rta is required for dimerization and DNA binding (20).
Another well-conserved region is at the C terminus and is rich in
acidic residues, which is characteristic of activation domains of many
transcriptional activators. It has been shown that this region of EBV
Rta is essential for transcriptional activation (20).
Therefore, the Rta homologues of gammaherpesviruses share similar amino
acid sequences and may also have similar functions in activating viral
and possibly cellular promoters. However, it is not clear whether the
Rta proteins are capable of substituting for each other to
transactivate virus-specific promoters. It has been shown that BHV-4
Rta could not transactivate the viral promoters that were activated by
either HVS or EBV Rta, nor could EBV Rta transactivate the viral
promoters that were activated by either BHV-4 or HVS Rta
(39). This specificity suggests that despite the
conservation of the functions in transactivation, virus- or host-specific interactions may be involved in mediating such functions.
MHV-68 Rta alone was sufficient to reactivate the virus in B cells.
Transfection of an Rta expression plasmid induced the expression of
early and late viral RNAs and proteins, activated lytic replication of
viral DNA, and led to the production and release of infectious viral
particles. So far, no other reports have demonstrated that Rta alone is
sufficient to drive the progression of the viral lytic cycle to the
production of infectious virions. In our time course experiments (Fig.
4), the lytic gene expression induced by Rta in S11E cells proceeded in
a cascade similar to that observed in de novo lytic infection of BHK-21
cells (Fig. 2). After Rta transfection, the early 2.6-kb TK transcript
was expressed to a high level at 12 h posttransfection, followed
by the late 0.9- and 3.6-kb M9 transcripts, peaking at 24 h
posttransfection (Fig. 4). The highest level of viral DNA was observed
at 24 h posttransfection (Fig. 5 and 6), followed by the maximal
accumulation of infectious particles in the supernatants occurring at
36 h posttransfection (Fig. 7). Nevertheless, we noticed that
there was significantly more intensive viral lytic replication in de novo-infected BHK-21 cells than in S11E cells transfected by
pcDNA3/MHV-68 rta. Differences in the replication levels
between the two cell types may be due to a suboptimal microenvironment
in B cells for viral lytic replication. This interpretation is
consistent with the predominantly latent nature of B-cell infection. We
also observed that one major lytic viral protein expressed in de
novo-infected BHK-21 cells was not seen in S11E cells transfected by
pcDNA3/MHV-68 rta. Because the antibody used for Western
analysis is polyclonal, the identity of this viral protein is not
clear. However, since there are infectious particles produced and
released into the medium, the viral protein not detected in Rta-induced
reactivation is unlikely to be essential for viral replication in B cells.
Based on our results that rta was expressed as an
immediate-early gene during de novo lytic infection and that
transfection of an Rta expression plasmid induced the viral lytic cycle
in latently infected B cells, we propose a working model for the functions of MHV-68 Rta. In this model, Rta is the central viral factor
determining lytic replication or latency of MHV-68. During de novo
infection of permissive cells such as BHK-21, Rta, presumably activated
by cooperation between a virion protein similar to HSV VP16 and
cellular transcription factors, is expressed to drive the viral lytic
cycle. In nonpermissive cells such as B cells, Rta expression is
blocked, which prevents initiation of the viral lytic cascade, leading
to latency. Sustained latency may require expression of viral latent
genes. However, in response to certain stimuli, Rta expression is
either activated or derepressed, and then latency is disrupted and the
virus undergoes reactivation. This model strongly points to regulation
of Rta expression governing the balance between viral latency and lytic
replication. Moreover, controlling the expression of Rta may allow us
to tip the balance between latency and lytic replication. Therefore,
MHV-68 offers a unique model system in which to study the consequences
of such a balance to viral pathogenesis.
| |
ACKNOWLEDGMENTS |
We thank Helen Brown, Tammy Rickabaugh, and Tonia Symensma
for critical comments and Wendy Aft for editing the manuscript.
This work is supported by the Frontiers of Science Award. T.-T.W. is
supported by a fellowship from Cancer Research Institute. E.J.U. is
supported by NIH grant AI37597. J.P.S. is a Royal Society University
Research Fellow.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular and Medical Pharmacology, University of California at Los
Angeles, Los Angeles, CA 90095-1735. Phone: (310) 794-5557. Fax: (310) 794-5123. E-mail: rsun{at}mednet.ucla.edu.
| |
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Abstract
Introduction
