Previous Article | Next Article 
Journal of Virology, March 1999, p. 2321-2332, Vol. 73, No. 3
Center for Immunology and Departments of
Pathology and Molecular Microbiology, Washington University School
of Medicine, St. Louis, Missouri 63110
Received 28 September 1998/Accepted 3 December 1998
The program(s) of gene expression operating during murine
gammaherpesvirus 68 ( Gammaherpesviruses are characterized
biologically by their association with tumors in immunosuppressed
hosts. The prototypic gammaherpesvirus 2, herpesvirus saimiri (HVS),
causes lymphomas in primates and can transform T lymphocytes (25,
31, 42, 48). Epstein-Barr virus (EBV) is associated with
lymphomas and nasopharyngeal carcinoma in humans (33, 58).
Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is
associated with Kaposi's sarcoma, body cavity-based lymphomas, and
Castleman's disease in humans (8, 11, 46, 65). Analysis of
transcripts expressed by these primate viruses in tumors and latently
infected cells has provided important information on both the
mechanisms of pathogenesis for these viruses and the cellular machinery
involved in host immune responses, cell cycle regulation, and cytokine
signaling. The species specificity of primate viruses such as EBV and
KSHV has, however, limited analysis of the role of these transcripts in vivo.
The availability of gammaherpesvirus 68 ( Two studies have suggested that B lymphocytes are the sole reservoir
within the hematopoietic compartment for Viruses, tissue culture, and mice.
Limiting-dilution analysis.
Assays for detection of latency
and preformed infectious RNA samples for RT-PCR.
Adult B-cell-deficient mice were
inoculated intraperitoneally (i.p.) with 106 PFU of RNA preparation and RT-PCR assays.
RNA was prepared by the
method of Chomczynski and Sacchi (12, 54). Briefly, spleen
or PEC samples were homogenized in 10 ml of GITC-phenol (mixed 1:0.1:1
with one of the following: [i] 4 M GITC-0.1 M
Primers for RT-PCR and determination of PCR sensitivity.
Primers were designed (Table 1) by using
vector NTI (Informax, Gaithersburg, Md.). In general we designed
primers that had a G+C content of approximately 50% so that a single
PCR program could be used for all primers. To determine the sensitivity
of our nested PCR, we prepared the
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Three Distinct Regions of the Murine Gammaherpesvirus 68 Genome
Are Transcriptionally Active in Latently Infected Mice
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
HV68) latency is undefined, as is the
relationship between HV68 latency and latency of primate
gammaherpesviruses. We used a nested reverse transcriptase PCR strategy
(sensitive to approximately one copy of HV68 genome for each genomic
region tested) to screen for the presence of viral transcripts in
latently infected mice. Based on the positions of known
latency-associated genes in other gammaherpesviruses, we screened for
the presence of transcripts corresponding to 11 open reading frames
(ORFs) in the HV68 genome in RNA from spleens and peritoneal cells
of latently infected B-cell-deficient (MuMT) mice which have been shown
contain high levels of reactivable latent HV68 (K. E. Weck, M. L. Barkon, L. I. Yoo, S. H. Speck, and H. W. Virgin, J. Virol. 70:6775-6780, 1996). To control for the
possible presence of viral lytic activity, we determined that RNA from
latently infected peritoneal and spleen cells contained few or no
detectable transcripts corresponding to seven ORFs known to encode
viral gene products associated with lytic replication. However, we did
detect low-level expression of transcripts arising from the region of
gene 50 (encoding the putative homolog of the Epstein-Barr virus BRLF1
transactivator) in peritoneal but not spleen cells. Latently infected
peritoneal cells consistently scored for expression of RNA derived from
4 of the 11 candidate latency-associated ORFs examined, including the
regions of ORF M2, ORF M11 (encoding v-bcl-2), gene 73 (a homolog of
the Kaposi's sarcoma-associated herpesvirus [human herpesvirus 8]
gene encoding latency-associated nuclear antigen), and gene 74 (encoding a G-protein coupled receptor homolog, v-GCR). Latently
infected spleen cells consistently scored positive for RNA derived from
3 of the 11 candidate latency-associated ORFs examined, including ORF
M2, ORF M3, and ORF M9. To further characterize transcription of these
candidate latency-associated ORFs, we examined their transcription in
lytically infected fibroblasts by Northern analysis. We detected
abundant transcription from regions of the genome containing ORF M3 and
ORF M9, as well as the known lytic-cycle genes. However,
transcription of ORF M2, ORF M11, gene 73, and gene 74 was barely
detectable in lytically infected fibroblasts, consistent with a role of
these viral genes during latent infection. We conclude that (i) we have
identified several candidate latency genes of murine HV68, (ii)
expression of genes during latency may be different in different
organs, consistent with multiple latency programs and/or multiple
cellular sites of latency, and (iii) regions of the viral genome
(v-bcl-2 gene, v-GCR gene, and gene 73) are transcribed during latency
with both HV68 and primate gammaherpesviruses. The implications of
these findings for replacing previous operational definitions of
HV68 latency with a molecular definition are discussed.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
HV68 or MHV68), a murine
virus well suited to pathogenesis studies, presents the opportunity to
evaluate the role of individual gammaherpesvirus genes in a model
amenable to both genetic and pathogenetic studies (76-78).
Analysis of the HV68 genome demonstrates that this virus is closely
related to primate gammaherpesviruses, including EBV, KSHV, and HVS
(21, 22, 76), but regions of the HV68 genome transcribed
during latency have not been defined. HV68 is a natural pathogen of
wild rodents (4, 44), capable of infecting both outbred and
inbred mice (5, 44, 56, 71). In one study, a significant
portion of mice infected with HV68 developed lymphoproliferative disorders. Treatment with cyclosporine increased the frequency of
lymphoproliferative disease (70). HV68 infects multiple organs of inbred mice and can establish a latent infection in the
spleen (5, 56, 71, 72, 77). Pending development of a
molecular definition of HV68 latency, we operationally define latency as the absence of preformed infectious virus, as measured by an
assay of defined sensitivity, and the capacity to reactivate virus
(77).
HV68 (72, 75).
In addition, a B-lymphoma cell line chronically infected with HV68
has been isolated from an infected mouse (74). However, the
issue of the cellular reservoir for latent virus within the lymphoid
organs remains unclear since subsequent analyses have demonstrated
efficient establishment of splenic latency in mice lacking mature B
cells (77) and persistence of HV68 DNA in lungs of
B-cell-deficient mice (69, 75). The presence of latent virus
in cells other than B cells was supported by a recent study suggesting
that epithelial cells are a site of HV68 persistence in lungs of
B-cell-deficient mice (69). Data on latency sites in vivo
therefore suggest that multiple cell types harbor latent virus.
Consistent with this, members of our group have found that latent
HV68 can be found in spleen, bone marrow, and peritoneal exudate
cells (PECs) (77, 78a) and identified macrophages as well as
B cells as sites of HV68 latency (78a). To further
understand HV68 latency and to generate a molecular, rather than
functional, definition of latency for HV68, we have performed
experiments to identify regions of the HV68 genome that are
transcribed in spleens and peritoneal cells from latently infected mice.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
HV68 was passaged and
assayed as described previously (77). Experiments were
performed with the HV68 WUMS clone (American Type Culture
Collection), which was doubly cloned by limiting dilution from a stock
originally supplied by Peter Doherty and Tony Nash. This isolate has
been sequenced in full (76). Plaque assays were performed
with NIH 3T12 cells (CCL 164; American Type Culture Collection).
HV68 was handled with strict biosafety level 2 precautions, as
HV68 infects human cells (reference 4 and unpublished observations). NIH 3T12 cells were maintained in
Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal
calf serum, 100 U of penicillin/ml, 100 mg of streptomycin/ml, 10 mM HEPES, and 2 mM L-glutamine (complete DMEM). Murine
embryonic fibroblasts (MEFs) were isolated and used as described
previously (77). Mice were housed and bred at the Washington
University School of Medicine at biosafety level 2 in accordance with
all federal and university regulations. Sentinel mice screened negative every 2 to 4 months for adventitious mouse pathogens. B-cell-deficient MuMT mice on the C57BL/6 background (C57BL/6J-Igh-6tm1Cgn)
were obtained from the Jackson Laboratory (Bar Harbor, Maine). B-cell-deficient mice were derived by interruption of the exon encoding
the transmembrane domain of immunoglobulin M and are deficient in
mature B cells (34).
HV68 were performed as previously reported
(77). Briefly, serial twofold dilutions (24 wells/dilution)
of stock HV68 or of infected mouse splenocytes were plated on
monolayers of 1 × 104 to 1.5 × 104
MEFs/well in 96-well tissue culture plates. MEFs were observed microscopically for viral cytopathic effect (CPE) for up to 4 weeks. To
differentiate between latently infected cells and preformed infectious
virus in cell samples, serial twofold dilutions of spleen cells or PECs
were plated before or after mechanical disruption of viable cells. For
mechanical disruption, cells were resuspended at a concentration of
1 × 106 to 5 × 106 cells/ml in
hypotonic 1/3× DMEM in the presence of 100 µl of 0.5-mm-diameter
zirconia-silica beads/ml and shaken twice for 1 min at high speed in a
Mini-Beadbeater-8 (Biospec Products, Bartlesville, Okla.). Published
studies have shown that mechanical disruption has at most a twofold
effect on the infectivity of preformed infectious HV68, while >99%
of cells are killed (77). Thus, mechanical disruption has
minimal effects on detection of preformed infectious virus in tissues
but efficiently eliminates latent cells. The limiting-dilution assay is
5- to 10-fold more sensitive than plaque assay on NIH 3T12 cells for
detecting preformed infectious HV68 (77, 78a).
HV68
in 0.5 ml of complete DMEM, and peritoneal cells and spleens were
harvested as previously described (28, 77). RNA used as a
negative control for reverse transcriptase (RT)-PCR assays was isolated
from uninfected thioglycolate-elicited PECs (52). Latent
peritoneal cell RNA was derived from nonelicited peritoneal cells
harvested from the following four groups of mice: (i) 7 mice 45 days
after infection, (ii) 9 mice 46 days after infection, (iii) 28 mice 42 days after infection, and (iv) 19 mice 42 days after infection. Spleen
RNA was harvested from mice 46 days after infection. Two separate pools
of spleen RNA were made and analyzed, each containing RNA from three
individual mouse spleens. RT-PCR was performed for every set of primers
on at least two different pools of peritoneal cell RNA and on both
pools of spleen RNA. RT-PCR data obtained from different pools of RNA
were consistent.
-mercaptoethanol-0.5% sarcosyl, [ii] 2 M sodium acetate [pH
4], and [iii] water-saturated phenol) and RNA was isolated. Spleens
were homogenized with a Tissue Tearor (Biospec Products), while PECs
were homogenized by repeated pipetting. Following initial RNA
preparation, samples were resuspended in 25 µl of DMPC-treated water
and treated for 15 min at 25°C with 2 µl of DNase I (2 U; Gibco
BRL, Grand Island, N.Y.) per the manufacturer's instructions.
DNase-treated RNA was resuspended in 1 ml of GITC-phenol and
reextracted by the same methods as described above. RNA was resuspended
in 50 to 100 µl of DMPC-treated water and quantitated, and the DNase
treatment was repeated. RNA (2 µg/experiment/primer set) was reverse
transcribed by using the Superscript preamplification system for
first-strand cDNA synthesis in a total volume of 20 µl (Gibco BRL),
and a no-RT control was carried out in parallel. Individual nested PCRs
were performed on 2 µl of the RT mixture in 1× reaction buffer
(Promega, Madison, Wis.) containing 2.5 mM MgCl2, a 0.2 mM
concentration of each nucleotide, a 0.15 µM concentration of each
primer (see Table 1 for primer sequences), and 1.25 U of Taq
DNA polymerase (Promega) in a final volume of 50 µl per PCR. Controls
for each PCR included water controls, no-RT controls, and multiple
samples containing 1 or 10 copies of the HV68 genome diluted in
0.1-mg/ml tRNA (Sigma, St. Louis, Mo.) combined with cDNA made by RT
treatment of RNA from uninfected mice. PCR was performed in a GeneAmp
PCR System 9600 (Perkin-Elmer, Norwalk, Conn.). The initial round of
PCR was performed with 25 cycles of 94°C for 1 min, 62°C for 1 min,
and 72°C for 1 min. Two microliters of the initial reaction mixture
was entered into the second round of each nested PCR. The second round
of PCR was performed under the same conditions as described above for
35 cycles. Both PCR programs began with an initial denaturation at 94°C for 2 min and concluded with a final hold at 72°C for 5 min. PCR products were stored at 4°C until separation by electrophoresis on a 2% agarose gel. PCR products were visualized by staining with
ethidium bromide.
HV68 genome as previously
described (76). The genome copy number was calculated
assuming a genome size of 150 kb and a molecular mass of 1.7 × 10
10 µg/molecule. Note that we assumed that the average
HV68 genome has approximately 150 kb, based on the unique sequence
of 118,237 bp plus about 30,000 bp (or ca. 26 copies) of the 1,213-bp
terminal repeats (76). While this is an arbitrary
assignment, the KSHV genome has about 30,000 bp of terminal repeat
sequence (ca. 35 to 45 copies of an 803-bp repeat) (37, 57,
59), and the HV68 genome carries on average at least 10 repeats (22). Our estimate of the genome size, and therefore
PCR assay sensitivity, is unlikely to be more than twofold off based on
the assumptions used to calculate the genome copy number. We prepared
stocks allowing us to add approximately 1 or approximately 10 copies of
the HV68 genome to samples containing cDNA made from naive mouse
tissue RNA, and we then ran these samples in parallel with cDNA samples from latently infected tissues. We selected nested primer pairs that
allowed consistent detection of one copy of the HV68 genome in a
background of cDNA (see Results and Table
2).
TABLE 1.
Primers used in this study
TABLE 2.
Detection of genome regions by PCR
and RT-PCRa
Isolation of RNA from lytically infected fibroblasts and Northern
blotting.
NIH 3T12 fibroblasts (2 × 107
cells/T175 flask) were pretreated with drugs as outlined below for
1 h and then infected at a multiplicity of infection of 7 in a
volume of 25 ml containing drugs, and flasks were incubated at 37°C
in a 5% CO2 atmosphere for 12 or 18 h prior to
harvest of RNA. Flasks were either left untreated or treated with drugs
as follows: (i) HV68 DNA synthesis was inhibited by adding
phosphonoacetic acid (PAA) at a final concentration of 200 µg/ml, and
(ii) protein synthesis was inhibited by adding a combination of
cycloheximide at a final concentration of 40 µM and anisomycin at a
final concentration of 10 µM. Total cellular RNA was harvested as
above and analyzed by Northern blot hybridization (60).
Probes were generated by cloning the PCR product of the outer sets of
primers for each genome region (Table 1). All probes were sequenced to
confirm their identities. In this way Northern probes encompassing the
region of the HV68 genome evaluated for transcription in latently
infected tissues were obtained. Blots were probed for rat cyclophilin
(13) to assess loading and RNA quality. All probes were
radiolabeled with the Megaprime DNA labeling system (Amersham,
Arlington Heights, Ill.) per the manufacturer's protocol. To provide
an internal control on each blot for differences in probe-specific
activity, we dotted 40 ng of HV68 genomic DNA prepared as described
previously (76) onto membranes prior to hybridization with
labeled probe.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
Selection of candidate regions of the genome to evaluate for
transcription during latent infection.
As there are currently no
tightly latent HV68-infected cell lines available, we evaluated
viral transcription in spleen and peritoneal cells isolated from
latently infected mice. Viral gene expression during
gammaherpesvirus latency has been thoroughly characterized
for EBV, and tumor- and latency-associated genes of KSHV and HVS
have also been identified (Fig. 1).
Although there are no clear homologs for many of these genes in
HV68, most of the tumor- or latency-associated genes of EBV, HVS,
and KSHV map either between the conserved regions of the viral genomes
or between KSHV and HVS genes 72 to 74 (Fig. 1), suggesting that the
equivalent regions of HV68 may also contain genes associated with
latent infection or tumor induction.
|
S below- the black rectangles. The identification and
characterization of EBV gene transcription in latently infected B cells
and EBV-associated tumors are reviewed in references
100 amino acids in length) identified in the
primary sequence of the HV68 genome (76), 40 (including
M7 and M8) have products that are homologous to tegument proteins,
structural proteins, or other proteins known or suspected to function
during the lytic cycle. Of these, we screened 8 for expression in
latently infected tissue (Tables 2 and
3). This afforded a direct assessment of
lytic gene transcription in latently infected tissue. Of the remaining
40 ORFs in HV68, 23 are homologous to ORFs in other gammaherpesviruses but are of unknown function. We did not examine these ORFs in this study, although they may encode proteins expressed during latency (62). Of the remaining 17 ORFs, we selected
11 to screen for expression in latently infected tissue (Tables 2 and
3). Four of these are in a cluster of genes {M11 (encoding a protein
containing a BH1 domain similar to bcl-2 family members, v-bcl-2), gene
72 (encoding a D-type cyclin, v-cyclin), gene 73 (homolog of the KSHV
latency-associated nuclear antigen [LANA] gene [32,
55]), and gene 74 (encoding a G-protein coupled receptor
homolog, v-GCR)} which are known to be, or suspected of being,
involved in KSHV or HVS latency and/or tumorigenesis (Fig. 1 and
Discussion). The other seven putative HV68 ORFs that we targeted
(ORFs M1, M2, M3, M4, M5, M6, and M9) do not have homologs in the known
gammaherpesviruses but are located in regions of the HV68 genome
positionally homologous to regions in other gammaherpesviruses that
contain genes expressed either in latently infected cell lines or in
tumors (Fig. 1) (76). Six other ORFs unique to HV68 (ORFs
M10a, M10b, M10c, M12, M13, and M14) span either the G+C-rich 100-bp
repeat region or the G+C-rich terminal repeats, making design of PCR
primers difficult, and thus we did not screen for the presence of
transcripts arising from these regions of the viral genome.
|
Development of sensitive nested RT-PCR assays for detection
of HV68 transcripts in tissue RNA.
To assess transcription of
candidate HV68 latency-associated genes, we developed sensitive
RT-PCR assays for detection of HV68 transcripts in RNA samples
from infected tissue. We designed primers (Table 1) meeting the
following criteria: (i) they are approximately 50% G+C, (ii) the outer
primers amplify a region 300 to 500 bp in length, (iii) independent
inner primers amplify a region 100 to 400 bp in length, and (iv)
amplified sequences are completely within ATG-initiated ORFs whose
products are at least 100 amino acids in length (76). These
criteria were selected to optimize RT-PCR sensitivity by amplifying
only short regions of RNA transcripts derived from regions containing
candidate genes. This approach, which does not provide information on
the direction of transcription or the structure of the RNA detected,
serves to identify regions of the genome that are transcriptionally active.
HV68 genome calculated to contain either 1 or 10 genome copies (see Materials and Methods). To measure sensitivity under
RT-PCR conditions, we performed all sensitivity assays in the presence
of cDNA derived by reverse transcription of RNA isolated from
uninfected mouse tissues (see Materials and Methods). Using this
approach we generated 19 nested PCR primer sets that reproducibly
detected 1 copy and 10 copies of the HV68 genome (Table 2). Note
that our false-positive rate (PCR products present after PCR of RNA
samples without added RT or using cDNA generated from uninfected mice)
was 4/900 PCRs, or 0.4% (Table 2). Three of four false-positives were
seen in RNA derived from peritoneal cells (no RT), a site of latency
that contains the highest frequency of HV68 genome-positive cells of
any tissue evaluated in our laboratory (78a). This raises
the possibility that these false positives may have been due to very
low levels of HV68 DNA in RNA preparations from peritoneal cells,
despite treatment with DNase prior to reverse transcription. To provide meaningful RT-PCR results we (i) performed multiple independent RT-PCRs
(usually four to eight per condition) within each experiment, (ii)
included data only from assays in which the sensitivity was 1 to 10 copies of genome, (iii) used cDNA derived from at least two independent
RT reactions from each RNA preparation, and (iv) analyzed RNA isolated
from multiple different groups of animals (Table 2; see also Materials
and Methods).
Minimal expression of lytic transcripts in tissues from latently
infected mice.
Previous studies have shown that HV68
efficiently establishes latency in the spleens of B-cell-deficient mice
after i.p. inoculation of HV68 (77). Peritoneal cells
were also selected for analysis in this study since it has been shown
that they are also tightly latent and regularly carry approximately
about 50- to 100-fold more cells that reactivate HV68 than spleen
cells (77, 78a). The definition of latency that members of
our group have used in earlier studies is based on the use of a
sensitive cocultivation assay that is approximately 5- to 10-fold more
sensitive than the standard plaque assay for detecting preformed
infectious HV68 in tissue samples (77, 78a). Latency is
defined as the presence of cells that can reactivate HV68 in the
absence of preformed infectious HV68. The presence of preformed
infectious HV68 is determined by the presence of virus after
mechanical disruption of live cells by the previously described
cocultivation assay (77). Mechanical disruption of cells
removes any contribution of reactivation of latent virus, since the
latter requires live cells. Importantly, this is an operational
definition of latency and has not been independently validated by
evaluating HV68 lytic gene transcription. For this reason we first
examined transcription from regions of the genome predicted or known to
encode lytic gene products in RNA from tissues operationally defined as
latently infected (i.e., no detectable preformed virus).
HV68. At this time
both peritoneal cells and spleen cells did not contain any detectable
preformed virus but did contain cells that reactivated HV68 (Fig.
2) (77, 78a). To determine
whether regions of the genome that encode lytic transcripts are
transcriptionally active in latent tissues, we used RT-PCR targeted to
genome regions (Tables 2 and 3) containing (i) two putative
immediate-early genes (K3 and gene 50; the latter is predicted to
encode a homolog of the EBV BRLF1 transactivator [27,
76]), (ii) three early genes (gene 6, encoding the ssDNA
binding protein [ssDNABP]; gene 8, encoding the DNA polymerase
[DNApol]; and M8, which is contained within the region encoding two
early mRNAs, including a spliced transcript [40]), and
(iii) three late genes (gene 25, encoding the major capsid
protein [MCP]; gene 7, encoding glycoprotein B [gB]
[68]); and M7, encoding glycoprotein 150 [gp150], a
known virion protein [67]). We did not detect
significant expression of seven of these lytic genes (K3, the ssDNABP
gene, the DNApol gene, M8, the MCP gene, the gB gene, or M7) in
either spleen or peritoneal cell RNA (Table 2). However, we did detect
at a low frequency transcripts derived from the gene 50 region in
peritoneal cells but not spleen cells (Table 2). The frequency of
detection did not meet our conservative criterion for selecting a gene
as a candidate latency gene (see below). Expression of a homolog of the
EBV BRLF1 transactivator might reflect abortive reactivation or the
presence of a very low level of RNA transcribed from the region of gene
50 during latency. Overall, the lack of significant transcription from
multiple regions of the genome containing lytic genes substantiates our
operational definition of latency and sets the stage for evaluation of
transcription from other regions of the genome in latently infected
tissue.
|
Identification of genome regions transcribed in latent
tissues.
Because we were successful in developing nested PCR
assays which exhibit single-copy sensitivity, and given the very low
false-positive rate of 0.4%, we elected the conservative criterion
that at least 50% of RT-PCR assays for transcription from a region of
the HV68 genome must be positive in order for us to make the
determination that a specific genome region contains a candidate
latency gene (Table 2). This conservative criterion will likely result
in us failing to call attention to bona fide candidate latent
transcripts, but we feel that this is preferable to falsely selecting
candidate latency genes for further analysis.
Analysis of HV68 gene expression in lytically infected
fibroblasts by Northern blotting.
The above-described analysis
identified ORFs M2, M3, M9, and M11 and genes 73 and 74 as regions of
the viral genome that are actively transcribed during latency and thus
as candidate latency genes. To further evaluate these candidates we
analyzed transcription of these putative viral genes in lytically
infected fibroblasts (Fig. 3 and Table
3). Our working assumption is that most viral genes expressed during
latent infection will either not be transcribed or be transcribed only
at a low level in lytically infected cells. To assess transcription of
the candidate latency genes in lytically infected fibroblasts, Northern
probes were derived by PCR amplification with the outer primers listed
in Table 1. Thus, in all cases, the probe used for Northern analysis
fully encompassed the region analyzed by RT-PCR (Tables 1 and 2). To
allow comparison of relative transcript levels of Northern blots
hybridized with different probes, we spotted 40 ng of denatured viral
genome onto each blot to provide an internal hybridization standard
(see Materials and Methods and the legend to Fig. 3). RNA for Northern
analysis was prepared from mock-infected cells and cells infected at a
multiplicity of infection of 7 and harvested at 12 h in the
presence or absence of either cycloheximide and anisomycin (will detect
transcription only of immediate-early genes) or PAA (will detect
transcription of immediate-early and early genes) and at 18 h
without drug treatment. These times were based on preliminary Northern
analyses demonstrating that HV68 late genes are expressed by 12 to
18 h in lytically infected fibroblasts (data not shown).
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
In this study we made the following important observations
(summarized in Table 3). First, we validated our operational definition of in vivo HV68 latency by demonstrating that a different program of
gene expression operates in latent tissues than is seen in lytically
infected fibroblasts. Second, we have identified three distinct regions
of the HV68 genome that are transcriptionally active during latency
(Fig. 1). The location of these regions correlates well with regions in
KSHV, EBV, and HVS that contain known latency and/or tumor-associated
genes, and some of the ORFs in these regions are homologous to known
latency- or tumor-associated genes of EBV, HVS, or KSHV. This provides
support for the use of HV68 in a small-animal model for evaluating
the molecular mechanisms underlying the latency of primate
gammaherpesviruses. Third, we present data consistent with the idea
that multiple programs of latency may operate in vivo, with different
sites showing different patterns of transcriptional activity. Fourth, we have shown that several of the candidate genes that appear to be
transcribed during latency are only weakly transcribed in lytically
infected fibroblasts.
Operational definitions of HV68 latency.
Analysis of
transcription during in vivo latency depends on a quantitative and
consistent operational definition of latency. Latency is the presence
of the HV68 viral genome in a reactivable form in the absence of
ongoing production of preformed infectious virus. A critical aspect of
this operational definition of latency is that the absence of preformed
virus (a negative result) is of necessity interpretable only when
information about the sensitivity of the assay used, and about the
survival of preformed virus in samples processed for analysis, is
provided. Several groups have operationally defined HV68 latency in
tissues by using different assays to detect preformed infectious virus.
Initial studies used a plaque assay to test for preformed infectious
virus, and investigators argued that virus detected as plaques by 5 to
7 days in an infectious-center assay reflected latently infected cells
reactivating HV68 since plaque assays failed to show infectious
virus (71). With this assay, infectious centers were
detected well after preformed infectious HV68 was cleared from
organs as measured by plaque assay, and several useful studies were
performed using this criterion (7, 20, 61, 71, 72, 75).
However, studies in the murine cytomegalovirus (MCMV) system from
multiple groups have shown that typical plaque assays for MCMV lack
sufficient sensitivity for preformed infectious virus in disrupted
organs to be a criterion for latency (36, 43, 53).
Similarly, two findings in the HV68 system invalidate plaque assay
results as a criterion for defining latency. First, using a
limiting-dilution assay, infectious HV68 has been detected in
tissues after clearance of infectious virus as measured by plaque assay
(77). Second, a recent study detected linear HV68 genomes
(demonstrated to be from lytic replication based on studies using a
viral DNA synthesis inhibitor in vivo) in the lung (69) when
standard assays for infectious virus fail to detect infectious virus
(71, 75, 77). These studies show that plaque assays for
HV68, as has been shown for MCMV, are not sufficiently sensitive to
provide a criterion for latency.
HV68
tRNA-like transcripts (6). While HV68 tRNA-positive cells
were observed in this study on day 21, the lack of preformed infectious
virus was addressed by the failure to demonstrate viral DNA by in situ
hybridization with a probe within the terminal repeats (a negative
result without quantitative data on sensitivity) and reliance on
previous studies showing by plaque assay that virus was cleared
(6). In another study, in situ hybridization for transcripts
from the glycoprotein H and thymidine kinase genes failed to
demonstrate lytically infected cells despite the fact that sensitive
PCR analysis demonstrated the presence of linear genomes and lytic
replication (69). While the sensitivity of in situ
hybridization for the viral terminal repeats versus transcripts from
lytic genes has not been addressed, these data provide a clear caution
against using the absence of a signal in in situ hybridization as a
criterion for the lack of lytic replication. In addition, the viral
tRNA-like genes of HV68 are abundantly transcribed during lytic
infection, and thus expression of the viral tRNA-like genes is not
specific for latency (6). Given the limitations of the
plaque assay (see above), the fact that in situ hybridization for viral
DNA is of unknown sensitivity in tissue sections, and the lack of
specificity of viral tRNA-like gene expression for latently infected
cells, expression of the viral tRNAs as a marker for latent cells will
require further substantiation.
Members of our group have used a different approach to assess organs
for preformed infectious virus (77). In this assay cells
from tissue are serially diluted onto permissive monolayers, and the
presence of infectious virus is detected by CPE that occurs over 2 to 4 weeks in culture (a significantly longer time than required for
detection of plaques formed by infectious centers [see above]). To
distinguish preformed infectious virus from latent virus, cells are
mechanically disrupted and diluted in parallel with live cells. This
assay is 5- to 10-fold more sensitive than the plaque assay for
detecting preformed infectious HV68 in tissue, and controls have
been performed to ensure that virus is not inactivated during tissue
preparation (77, 78a). The major advantage of this assay is
that it affords a direct comparison of the presence of preformed
infectious virus and virus reactivation from latency, since the same
assay with a defined sensitivity is used for both analyses. Thus, the
detection of viral CPE upon plating live cells in the absence of viral
CPE upon plating mechanically disrupted cells unambiguously
demonstrates the presence of virus reactivation from latency. Using
this assay, we have shown that HV68 establishes latency in the
absence of mature B cells (Fig. 2) (77), a finding confirmed
and extended in subsequent studies from other groups (69,
75). Notwithstanding the sensitivity of this assay and that of
others, such as PCR detection of linear genomes (69), all of
the criteria for latency used to date depend on the sensitivity of
assays to detect a negative result (no preformed virus, no lytic
transcripts by in situ hybridization, and no linear genomes), and a
positive molecular definition of HV68 latency has been lacking.
Molecular definition of HV68 latency.
Based on the
limitations of the above-described assays, we embarked on generating a
molecular definition of HV68 latency. This study represents the
first step in this direction. We felt that the strictest criteria that
could apply to defining HV68 latency were (i) the absence of
preformed infectious virus as determined by the most sensitive assay
currently available for detecting infectious HV68 in tissue
(limiting-dilution analysis [77]), (ii) the absence of
lytic gene transcription from multiple regions of the viral genome in
sensitive nested RT-PCR assays, and (iii) the identification of genome
regions preferentially expressed in latent tissues as opposed to
lytically infected cells.
HV68 latency. We believe
that we have detected true latency in vivo for several reasons. First,
minimal transcription from multiple regions of the genome containing
known lytic genes was detected in latent tissues by nested PCR assays
with one-copy sensitivity (Tables 2 and 3 and Fig. 1). While the
absence of transcription of a single lytic gene might be ignored based
on the idea that there could be a problem with the assay, we believe
that lack of transcription of seven different lytic genes strongly
argues against the presence of significant lytic gene expression in the
latently infected PEC and splenocyte populations. In contrast to
results obtained by RT-PCR with RNA isolated from latent tissues,
transcripts from these lytic genes was readily detected by Northern
blot analysis of RNA isolated from lytically infected fibroblasts (Fig.
3). Notably, transcripts from several genome regions (gene 73, gene 74, M11, M2) were detected in latent tissues (Table 2), despite the fact
that these regions were inefficiently transcribed in lytically infected
fibroblasts (Fig. 3). In addition, we identified transcription in
latent tissue from two regions of the HV68 genome that contain genes
(gene 73 and M11) homologous to genes transcribed during either EBV or
KSHV latency (see below). Together, these data strongly argue that
HV68 has at least one latent program of gene expression that is
distinct from the lytic program, and they provide for the first time a
positive criterion (the presence of transcription from genome regions
containing M2, M11, gene 73, and gene 74) for detecting latency.
We also detected transcription from regions of the genome encoding the
M3 and M9 ORFs in latently infected tissues (Tables 2 and 3). However,
in contrast to the situation with M2, M11, gene 73, and gene 74, the M3
and M9 genome regions are abundantly transcribed during lytic
infection. This clouds selection of M3 and M9 as candidate latent
genes. Notably, in EBV latent transcripts span multiple lytic genes,
with the latency-associated antigens translated from mRNAs in which the
regions containing the lytic-cycle genes have been spliced out. Thus,
it is possible that latent transcripts exist for HV68 that span
lytic genes contained in the M3 and M9 regions. This possibility is
supported by preliminary data showing that the M3 gene encodes an
abundant protein that is secreted from lytically infected cells
(75a). Further analysis of possible latent genes in the
regions of the M3 and M9 ORFs will therefore require definition of
latent transcript structure.
We were interested to see that the pattern of gene regions transcribed
to detectable levels in latent tissue differed for peritoneal cells and
spleens (Table 2). These apparent differences may be related to
different loads of latent cells or to differences in efficiency of RT
for specific mRNAs in RNA derived from different organs. An alternate
possibility is that HV68 has multiple latent gene programs (as has
been demonstrated for EBV) or that multiple different cell types, each
having a different gene program, carry HV68 during latency.
Interestingly, data for latency in both B cells and an additional cell
type (not B cells) in the lung have been presented (69, 72,
77), and members of our group have obtained data consistent with
HV68 latency in both B cells and macrophages (78a). These
studies support the idea that there are multiple sites of HV68
latency, and they support the need for evaluation of HV68 latent
gene transcription in different cell types in vivo.
ORFs in regions of the viral genome actively transcribed in latent tissue. Several of the candidate latency-associated genes (M2, M3, and M9) encode putative products with no clear homologs in the database (as determined by advanced Blast search of the nonredundant database at in September 1998). These regions are particularly interesting because they may encode proteins with novel functions that are important for persistence of viral infection in the face of active immunity. However, three of the candidate latency-associated genes are predicted to encode proteins homologous to viral antigens encoded by EBV, KSHV, and/or HVS that have been studied in some detail.
v-bcl-2.
Data presented here demonstrate that the region of
the HV68 genome containing M11, predicted to encode a protein
homologous to proteins of the bcl-2 family that regulate cell death, is
transcriptionally active during latency. The putative HV68 ORF
M11-encoded v-bcl-2 protein is homologous to the v-bcl-2 proteins of
EBV (BHRF1), KSHV (encoded by gene 16), and HVS (encoded by gene 16)
(76). There has been consistent agreement from a number of
studies that the gammaherpesvirus v-bcl-2 proteins inhibit apoptosis
(10, 15, 23, 29, 49, 63, 73). However, the function of the
v-bcl-2 proteins during viral infection is not clear, since the BHRF1
protein is not required for either viral replication or B-cell
transformation in vitro (39, 41). There are four recognized domains that are conserved between different bcl-2 family
members (BH1 to BH4). The HV68 v-bcl-2 protein has a
recognizable BH1-like domain but differs significantly from host and
v-bcl-2 family members in regions corresponding to BH2 to BH4 (10,
76). In addition to the BH1 domain, HV68 v-bcl-2 shares with
the EBV, KSHV, and HVS proteins a hydrophobic C-terminal domain
potentially involved in membrane localization. The preservation of
these structural motifs across gammaherpesvirus v-bcl-2 molecules
argues that these regions are functionally important.
HV68, minimal transcription of the M11 ORF, encoding v-bcl-2, was
detected in lytically infected fibroblasts, while RT-PCR detected
transcription of this region in latent tissue. While low levels of
expression of HV68 v-bcl-2 may be critical to lytic replication, our
data suggest that HV68 v-bcl-2 may play an important role during latency.
Gene 73 (encoding LANA).
Gene 73 is shared among
HV68, KSHV, and HVS. The KSHV gene 73 protein (LANA is expressed in
spindle cells of Kaposi's sarcoma [9, 32, 55]). It is
important to note that the amino acid homology among the products of
gene 73 of HV68, HVS, and KSHV is weaker than the homology seen
between the majority of lytic genes conserved in these viruses
(76). In particular, HV68 gene 73 lacks a long complex
repeat present (albeit with different structures and differing lengths)
in both KSHV and HVS. The function of this repeat is not known. KSHV
gene 73 and the KSHV v-cyclin gene are transcribed from a single
promoter region as a bicistronic message and a shorter spliced
form encoding v-cyclin (17). Thus, there is
coregulation of the transcription of the KSHV v-cyclin gene and gene
73, suggesting that these genes play important roles in the same part
of the viral life cycle. There is some upregulation of the KSHV
v-cyclin-specific mRNA by treatment with phorbol esters (17)
(perhaps consistent with a role for KSHV v-cyclin in lytic infection),
but data for expression of both the KSHV v-cyclin gene and KSHV gene 73 in tumor cells is strong (9, 14, 32, 55). Thus, KSHV
gene 73 and the KSHV v-cyclin gene, which are derived from the same
promoter, are clear latent genes. The situation for HV68 appears to
be different in two respects. The HV68 v-cyclin gene is
abundantly expressed in lytically infected fibroblasts (75b), while we detected little or no transcription from the region of gene 73 in lytically infected fibroblasts (Fig. 3). Thus,
there is independent regulation of HV68 gene 73 and the HV68
v-cyclin gene in lytically infected cells. The genome structure of
HV68, which has the v-bcl-2-encoding M11 gene interposed in the
opposite orientation between gene 73 and the v-cyclin gene, provides a
further basis for believing that the HV68 v-cyclin gene and gene 73 may be independently regulated (76). In addition, the RT-PCR
analysis of transcripts corresponding to the HV68 v-cyclin gene
region did not meet our conservative criteria for designating the
v-cyclin gene a candidate latency gene (Table 2), while gene 73 did
meet these criteria. This may reflect a preferential role of the
HV68 v-cyclin gene in lytic infection, although inefficient
detection of HV68 v-cyclin mRNA in latent tissues could be due to
poor efficiency of reverse transcription of this mRNA. Further studies
evaluating the role of the HV68 v-cyclin gene and HV68 gene 73 during latency and lytic infection will be needed to determine the
contribution of the viral gene products to latency and virus replication.
Gene 74 (v-GCR).
Previous studies have shown that the KSHV
interleukin-8 receptor-like antigen encoded by gene 74 (v-GCR
[59]) is a constitutively signaling receptor linked to
cell proliferation (1, 3) and is expressed in tumors
(9, 26). Transcriptional mapping in a KSHV-infected B-cell
line is consistent with the expression of the v-GCR gene as both a
latent and lytic transcript (62), although in another study
abundant expression of an mRNA encoding the KSHV-v-GCR in phorbol
ester-treated cells was noted (17). These data are in
contrast to the situation with HV68. We found minimal transcription
from the v-GCR gene region in lytically infected cells, but we did
detect expression from this region in latently infected peritoneal
cells. Thus, in contrast to the situation in KSHV, it seems likely that
the HV68 v-GCR plays an important role during latent but not lytic infection.
HV68 as a model for analysis of in vivo gammaherpesvirus
latency.
One of the fundamentally important issues confronted by
those using animal models of viral infection is how well the model relates to human disease processes. The HV68 model presents certain very compelling advantages for the study of the basic biology of
gammaherpesvirus latency. These include the close genetic relatedness in certain regions of the HV68 genome to other primate and human gammaherpesviruses, the availability of a manipulable animal model, and
the ease with which mutants of HV68 can be made (reference 64 and unpublished data). However, it is still
unclear whether HV68 will serve as a useful model at the level of
molecular mechanisms. This study presents the first molecular data
which afford an initial assessment of the issue of whether HV68
serves as a model for latency of EBV and KSHV.
HV68 is distinct in some respects from other
gammaherpesviruses, such as EBV and KSHV, insofar as latency- and
tumor-associated gene expression is concerned. For example, none
of the EBNA genes of EBV are clearly represented in the HV68 genome
(76). In addition, known or candidate latency- or
tumor-associated KSHV genes (such as the viral IRF gene [24,
45]; the K12 gene, encoding kaposin protein [47, 66,
80]; and K1 [38]) are not easily recognized
in the HV68 genomic sequence (76). HV68 contains no
obvious homologs of the HVS transforming STP and Tip genes (16,
19, 25, 31, 76) or ORF 14 (18). This argues either
that HV68 uses distinct pathways for tumor generation and/or latency
or that HV68 has arrived at different strategies for addressing the
problems addressed by the EBNA genes or certain KSHV and HVS genes.
Similarly, there are no obvious homologs in KSHV, EBV, or HVS of the
HV68 M2, M3, and M9 genes. In addition, preliminary analysis
suggests that even genes that may be held in common between the latency
programs of KSHV and HV68 (gene 73 and the v-GCR gene) or EBV and
HV68 (the v-bcl-2 gene) may be regulated differently (see above).
Despite these differences, the data presented here provide a compelling
argument for the use of HV68 to assess the role of certain
gammaherpesvirus genes in latency and infection in vivo. These include
the v-bcl-2 gene (M11), gene 73, and gene 74. The regions of the viral
genome containing these genes are transcribed in latent tissues,
tumors, and tumor cell lines infected with HV68 and either both EBV
and KSHV or KSHV alone. We believe that this argues strongly for the
validity of HV68 for assessing the function of certain genes in
gammaherpesvirus biology.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by a grant to H.W.V. from the National Cancer Institute (RO1 CA74730). S.H.S. was supported by NIH RO1 grants CA43143, CA52004, CA58524, and CA74730 from the National Cancer Institute. R.M.P. was supported by NIH grants GM07200 and AI07163.
We acknowledge helpful discussions from members of the Speck and Virgin labs, as well as discussions that occurred during lab meetings shared with David Leib.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Center for Immunology and Departments of Pathology and Molecular Microbiology, Washington University School of Medicine, Box 8118, 660 S. Euclid Ave., St. Louis, MO 63110. Phone: (314) 362-0367 (S.H.S.) and (314) 362-9223 (H.W.V.). Fax: (314) 362-4096. E-mail: virgin{at}immunology.wustl.edu (H.W.V.) and speck{at}pathology.wustl.edu (S.H.S.).
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
| 1. | Arvanitakis, L., E. Geras-Raaka, A. Varma, M. C. Gershengorn, and E. Cesarman. 1997. Human herpesvirus KSHV encodes a constitutively active G-protein-coupled receptor linked to cell proliferation. Nature 385:347-350[Medline]. |
| 2. |
Austin, P. J.,
E. Flemington,
C. N. Yandava,
J. L. Strominger, and S. H. Speck.
1988.
Complex transcription of the Epstein-Barr virus BamHI fragment H rightward open reading frame 1 (BHRF1) in latently and lytically infected B lymphocytes.
Proc. Natl. Acad. Sci. USA
85:3678-3682 |
| 3. | Bais, C., B. Santomasso, O. Coso, L. Arvanitakis, E. G. Raaka, J. S. Gutkind, A. S. Asch, E. Cesarman, M. C. Gershengorn, and E. A. Mesri. 1998. G-protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator. Nature 391:86-89[Medline]. (Erratum, 392:210.) |
| 4. | Blaskovic, D., M. Stancekova, J. Svobodova, and J. Mistrikova. 1980. Isolation of five strains of herpesviruses from two species of free living small rodents. Acta Virol. 24:468[Medline]. |
| 5. | Blaskovic, D., D. Stanekova, and J. Rajcani. 1984. Experimental pathogenesis of murine herpesvirus in newborn mice. Acta Virol. 28:225-231[Medline]. |
| 6. | Bowden, R. J., J. P. Simas, A. J. Davis, and S. Efstathiou. 1997. Murine gammaherpesvirus 68 encodes tRNA-like sequences which are expressed during latency. J. Gen. Virol. 78:1675-1687[Abstract]. |
| 7. |
Cardin, R. D.,
J. W. Brooks,
S. R. Sarawar, and P. C. Doherty.
1996.
Progressive loss of CD8+ T cell-mediated control of a gamma-herpesvirus in the absence of CD4+ T cells.
J. Exp. Med.
184:863-871 |
| 8. |
Cesarman, E.,
Y. Chang,
P. S. Moore,
J. W. Said, and D. M. Knowles.
1995.
Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas.
N. Engl. J. Med.
332:1186-1191 |
| 9. | Cesarman, E., R. G. Nador, F. Bai, R. A. Bohenzky, J. J. Russo, P. S. Moore, Y. Chang, and D. M. Knowles. 1996. Kaposi's sarcoma-associated herpesvirus contains G protein-coupled receptor and cyclin D homologs which are expressed in Kaposi's sarcoma and malignant lymphoma. J. Virol. 70:8218-8223[Abstract]. |
| 10. |
Chang, E. H. Y.,
J. Nicholas,
D. S. Bellows,
G. S. Hayward,
H.-G. Guo,
M. S. Reitz, and J. M. Hardwick.
1997.
A bcl-2 homolog encoded by Kaposi's sarcoma-associated virus, human herpesvirus 8, inhibits apoptosis but does not heterodimerize with Bax or Bak.
Proc. Natl. Acad. Sci. USA
94:690-694 |
| 11. |
Chang, Y.,
E. Cesarman,
M. S. Pessin,
F. Lee,
J. Culpepper,
D. M. Knowles, and P. S. Moore.
1994.
Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.
Science
266:1865-1869 |
| 12. | Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159[Medline]. |
| 12a. | Clambey, E. C., H. W. Virgin, and S. H. Speck. Unpublished observation. |
| 13. | Danielson, P. E., S. Forss-Petter, M. A. Brow, L. Calavetta, J. Douglass, R. J. Milner, and J. G. Sutcliffe. 1988. p1B15: a cDNA clone of the rat mRNA encoding cyclophilin. DNA 7:261-267[Medline]. |
| 14. |
Davis, M. A.,
M. A. Sturzl,
C. Blasig,
A. Schreier,
H. G. Guo,
M. Reitz,
S. R. Opalenik, and P. J. Browning.
1997.
Expression of human herpesvirus 8-encoded cyclin D in Kaposi's sarcoma spindle cells.
J. Natl. Cancer Inst.
89:1868-1874 |
| 15. | Dawson, C. W., A. G. Eliopoulos, J. Dawson, and L. S. Young. 1995. BHRF1, a viral homologue of the Bcl-2 oncogene, disturbs epithelial cell differentiation. Oncogene 10:69-77[Medline]. |
| 16. |
Desrosiers, R. C.,
A. Bakker,
J. Kamine,
L. A. Falk,
R. D. Hunt, and N. W. King.
1985.
A region of the herpesvirus saimiri genome required for oncogenicity.
Science
228:184-187 |
| 17. |
Dittmer, D.,
M. Lagunoff,
R. Renne,
K. Staskus,
A. Haase, and D. Ganem.
1998.
A cluster of latently expressed genes in Kaposi's sarcoma-associated herpesvirus.
J. Virol.
72:8309-8315 |
| 18. |
Duboise, M.,
J. Guo,
S. Czajak,
H. Lee,
R. Veazey,
R. C. Desrosiers, and J. U. Jung.
1998.
A role for herpesvirus saimiri orf14 in transformation and persistent infection.
J. Virol.
72:6770-6776 |
| 19. |
Duboise, S. M.,
J. Guo,
S. Czajak,
R. C. Desrosiers, and J. U. Jung.
1998.
STP and Tip are essential for herpesvirus saimiri oncogenicity.
J. Virol.
72:1308-1313 |
| 20. | Dutia, B. M., C. J. Clarke, D. J. Allen, and A. A. Nash. 1997. Pathological changes in the spleens of gamma interferon receptor-deficient mice infected with murine gammaherpesvirus: a role for CD8 T cells. J. Virol. 71:4278-4283[Abstract]. |
| 21. |
Efstathiou, S.,
Y. M. Ho,
S. Hall,
C. J. Styles,
S. D. Scott, and U. A. Gompels.
1990.
Murine herpesvirus 68 is genetically related to the gammaherpesviruses Epstein-Barr virus and herpesvirus saimiri.
J. Gen. Virol.
71:1365-1372 |
| 22. |
Efstathiou, S.,
Y. M. Ho, and A. C. Minson.
1990.
Cloning and molecular characterization of the murine herpesvirus 68 genome.
J. Gen. Virol.
71:1355-1364 |
| 23. | Foghsgaard, L., and M. Jaattela. 1997. The ability of BHRF1 to inhibit apoptosis is dependent on stimulus and cell type. J. Virol. 71:7509-7517[Abstract]. |
| 24. | Gao, S. J., C. Boshoff, S. Jayachandra, R. A. Weiss, Y. Chang, and P. S. Moore. 1997. KSHV ORF K9 (vIRF) is an oncogene which inhibits the interferon signaling pathway. Oncogene 15:1979-1985[Medline]. |
| 25. |
Geck, P.,
S. A. Whitaker,
M. M. Medveczky, and P. G. Medveczky.
1990.
Expression of collagenlike sequences by a tumor virus, herpesvirus saimiri.
J. Virol.
64:3509-3515 |
| 26. | Guo, H. G., P. Browning, J. Nicholas, G. S. Hayward, E. Tschachler, Y. W. Jiang, M. Sadowska, M. Raffeld, S. Colombini, R. C. Gallo, and M. S. J. Reitz. 1997. Characterization of a chemokine receptor-related gene in human herpesvirus 8 and its expression in Kaposi's sarcoma. Virology 228:371-378[Medline]. |
| 27. |
Hardwick, J. M.,
P. M. Lieberman, and S. D. Hayward.
1988.
A new Epstein-Barr virus transactivator, R, induces expression of a cytoplasmic early antigen.
J. Virol.
62:2274-2284 |
| 28. | Heise, M. T., and H. W. Virgin, IV. 1995. The T-cell-independent role of gamma interferon and tumor necrosis factor alpha in macrophage activation during murine cytomegalovirus and herpes simplex virus infections. J. Virol. 69:904-909[Abstract]. |
| 29. |
Henderson, S.,
D. Huen,
M. Rowe,
C. Dawson,
G. Johnson, and A. Rickinson.
1993.
Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death.
Proc. Natl. Acad. Sci. USA
90:8479-8483 |
| 30. |
Jung, J. U.,
M. Stäger, and R. C. Desrosiers.
1994.
Virus-encoded cyclin.
Mol. Cell. Biol.
14:7235-7244 |
| 31. |
Jung, J. U.,
J. J. Trimble,
N. W. King,
B. Biesinger,
B. W. Fleckenstein, and R. C. Desrosiers.
1991.
Identification of transforming genes of subgroup A and C strains of herpesvirus saimiri.
Proc. Natl. Acad. Sci. USA
88:7051-7055 |
| 32. | Kedes, D. H., M. Lagunoff, R. Renne, and D. Ganem. 1997. Identification of the gene encoding the major latency-associated nuclear antigen of the Kaposi's sarcoma-associated herpesvirus. J. Clin. Investig. 100:2606-2610[Medline]. |
| 33. | Kieff, E. 1996. Epstein-Barr virus and its replication, p. 2343-2396. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology. Lippincott-Raven, Philadelphia, Pa. |
| 34. | Kitamura, D., J. Roes, R. Kuhn, and K. Rajewsky. 1991. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature 350:423-426[Medline]. |
| 35. |
Kraft, M. S.,
G. Henning,
H. Fickenscher,
D. Lengenfelder,
J. Tschopp,
B. Fleckenstein, and E. Meinl.
1998.
Herpesvirus saimiri transforms human T-cell clones to stable growth without inducing resistance to apoptosis.
J. Virol.
72:3138-3145 |
| 36. | Kurz, S., H.-P. Steffens, A. Mayer, J. R. Harris, and M. J. Reddehase. 1997. Latency versus persistence or intermittent recurrences: evidence for a latent state of murine cytomegalovirus in the lungs. J. Virol. 71:2980-2987[Abstract]. |
| 37. | Lagunoff, M., and D. Ganem. 1997. The structure and coding organization of the genomic termini of Kaposi's sarcoma-associated herpesvirus. Virology 236:147-154[Medline]. |
| 38. | Lee, H., R. Veazey, K. Williams, M. Li, J. Guo, F. Neipel, B. Fleckenstein, A. Lackner, R. C. Desrosiers, and J. U. Jung. 1998. Deregulation of cell growth by the K1 gene of Kaposi's sarcoma-associated herpesvirus. Nat. Med. 4:435-440[Medline]. |
| 39. |
Lee, M.-A., and J. L. Yates.
1992.
BHRF1 of Epstein-Barr virus, which is homologous to human proto-oncogene bcl2, is not essential for transformation of B cells or for virus replication in vitro.
J. Virol.
66:1899-1906 |
| 39a. | Liu, C., H. W. Virgin IV, and S. H. Speck. Unpublished observation. |
| 40. | Mackett, M., J. P. Stewart, S. D. V. Pepper, M. Chee, S. Efstathiou, A. Nash, and J. R. Arrand. 1997. Genetic content and preliminary transcriptional analysis of a representative region of murine gammaherpesvirus 68. J. Gen. Virol. 78:1425-1433[Abstract]. |
| 41. |
Marchini, A.,
B. Tomkinson,
J. I. Cohen, and E. Kieff.
1991.
BHRF1, the Epstein-Barr virus gene with homology to Bcl2, is dispensable for B-lymphocyte transformation and virus replication.
J. Virol.
65:5991-6000 |
| 42. | Medveczky, M., P. Geck, J. L. Sullivan, D. Serbousek, J. Y. Djeu, and P. G. Medveczky. 1993. IL-2 independent growth and cytotoxicity of herpesvirus saimiri-infected human CD8 cells and involvement of two open reading frame sequences of the virus. Virology 196:402-412[Medline]. |
| 43. |
Mercer, J. A.,
C. A. Wiley, and D. H. Spector.
1988.
Pathogenesis of murine cytomegalovirus infection: identification of infected cells in the spleen during acute and latent infections.
J. Virol.
62:987-997 |
| 44. | Mistrikova, J., and D. Blaskovic. 1985. Ecology of the murine alphaherpesvirus and its isolation from lungs of rodents in cell culture. Acta Virol. 29:312-317[Medline]. |
| 45. |
Moore, P. S.,
C. Boshoff,
R. A. Weiss, and Y. Chang.
1996.
Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV.
Science
274:1739-1744 |
| 46. |
Moore, P. S., and Y. Chang.
1995.
Detection of herpesvirus-like DNA sequences in Kaposi's sarcoma in patients with and without HIV infection.
N. Engl. J. Med.
332:1181-1185 |
| 47. |
Muralidhar, S.,
A. M. Pumfery,
M. Hassani,
M. R. Sadaie,
N. Azumi,
M. Kishishita,
J. N. Brady,
J. Doniger,
P. Medveczky, and L. J. Rosenthal.
1998.
Identification of kaposin (open reading frame K12) as a human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) transforming gene.
J. Virol.
72:4980-4988 |
| 48. |
Murthy, S. C. S.,
J. J. Trimble, and R. C. Desrosiers.
1989.
Deletion mutants of herpesvirus saimiri define an open reading frame necessary for transformation.
J. Virol.
63:3307-3314 |
| 49. | Nava, V. E., E. H.-Y. Cheng, M. Veliuona, S. Zou, R. J. Clem, M. L. Mayer, and J. M. Hardwick. 1997. Herpesvirus saimiri encodes a functional homolog of the human bcl-2 oncogene. J. Virol. 71:4118-4122[Abstract]. |
| 50. |
Neilan, J. G.,
Z. Lu,
C. L. Afonso,
G. F. Kutish,
M. D. Sussman, and D. L. Rock.
1993.
An African swine fever virus gene with similarity to the proto-oncogene bcl-2 and the Epstein-Barr virus gene BHRF1.
J. Virol.
67:4391-4394 |
| 51. | Pearson, G. R., J. Luka, L. Petti, J. Sample, M. Birkenbach, D. Braun, and E. Kieff. 1987. Identification of an Epstein-Barr virus early gene encoding a second component of the restricted early antigen complex. Virology 160:151-161[Medline]. |
| 52. | Pollock, J. L., R. M. Presti, S. Paetzold, and H. W. Virgin. 1997. Latent murine cytomegalovirus infection in macrophages. Virology 227:168-179[Medline]. |
| 53. | Pollock, J. L., and H. W. Virgin, IV. 1995. Latency, without persistence, of murine cytomegalovirus in spleen and kidney. J. Virol. 69:1762-1768[Abstract]. |
| 54. | Puglielli, M. T., M. Woisetschlaeger, and S. H. Speck. 1996. oriP is essential for EBNA gene promoter activity in Epstein-Barr virus-immortalized lymphoblastoid cell lines. J. Virol. 70:5758-5768[Abstract]. |
| 55. | Rainbow, L., G. M. Platt, G. R. Simpson, R. Sarid, S.-J. Gao, H. Stoiber, C. S. Herrington, P. S. Moore, and T. F. Schulz. 1997. The 222- to 234-kilodalton latent nuclear protein (LNA) of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) is encoded by orf73 and is a component of the latency-associated nuclear antigen. J. Virol. 71:5915-5921[Abstract]. |
| 56. | Rajcani, J., D. Blaskovic, J. Svobodova, F. Ciampor, D. Huckova, and D. Stanekova. 1985. Pathogenesis of acute and persistent murine herpesvirus infection in mice. Acta Virol. 29:51-60[Medline]. |
| 57. | Renne, R., M. Lagunoff, W. Zhong, and D. Ganem. 1996. The size and conformation of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) DNA in infected cells and virions. J. Virol. 70:8151-8154[Abstract]. |
| 58. | Rickinson, A. B., and E. Kieff. 1996. Epstein-Barr virus, p. 2397-2446. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology. Lippincott-Raven, Philadelphia, Pa. |
| 59. |
Russo, J. J.,
R. A. Bohenzky,
M.-C. Chien,
J. Chen,
M. Yan,
D. Maddalena,
J. P. Parry,
D. Peruzzi,
I. S. Edelman,
Y. Chang, and P. S. Moore.
1996.
Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8).
Proc. Natl. Acad. Sci. USA
93:14862-14867 |
| 60. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 61. | Sarawar, S. R., R. D. Cardin, J. W. Brooks, M. Mehrpooya, A.-M. Hamilton-Easton, X. Y. Mo, and P. C. Doherty. 1997. Gamma interferon is not essential for recovery from acute infection with murine gammaherpesvirus 68. J. Virol. 71:3916-3921[Abstract]. |
| 62. |
Sarid, R.,
O. Flore,
R. A. Bohenzky,
Y. Chang, and P. S. Moore.
1998.
Transcription mapping of the Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) genome in a body cavity-based lymphoma cell line (BC-1).
J. Virol.
72:1005-1012 |
| 63. | Sarid, R., T. Sato, R. A. Bohenzky, J. J. Russo, and Y. Chang. 1997. Kaposi's sarcoma-associated herpesvirus encodes a functional bcl-2 homologue. Nat. Med. 3:293-298[Medline]. |
| 64. | Simas, J. P., R. J. Bowden, V. Paige, and S. Efstathiou. 1998. Four tRNA-like sequences and a serpin homologue encoded by murine gammaherpesvirus 68 are dispensable for lytic replication in vitro and latency in vivo. J. Gen. Virol. 79:149-153[Abstract]. |
| 65. |
Soulier, J.,
L. Grollet,
E. Oksenhendler,
P. Cacoub,
D. Cazals-Hatem,
P. Babinet,
M.-F. d'Agay,
J.-P. Clauvel,
M. Raphael,
L. Degos, and F. Signaux.
1995.
Kaposi's sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman's disease.
Blood
86:1276-1280 |
| 66. | Staskus, K. A., W. Zhong, K. Gebhard, B. Herndier, H. Wang, R. Renne, J. Beneke, J. Pudney, D. J. Anderson, D. Ganem, and A. T. Haase. 1997. Kaposi's sarcoma-associated herpesvirus gene expression in endothelial (spindle) tumor cells. J. Virol. 71:715-719[Abstract]. |
| 67. | Stewart, J. P., N. J. Janjua, S. D. V. Pepper, G. Bennion, M. Mackett, T. Allen, A. A. Nash, and J. R. Arrand. 1996. Identification and characterization of murine gammaherpesvirus 68 gp150: a virion membrane glycoprotein. J. Virol. 70:3528-3535[Abstract]. |
| 68. |
Stewart, J. P.,
N. J. Janjua,
N. P. Sunil-Chandra,
A. A. Nash, and J. R. Arrand.
1994.
Characterization of murine gammaherpesvirus 68 glycoprotein B (gB) homolog: similarity to Epstein-Barr virus gB (gp110).
J. Virol.
68:6496-6504 |
| 69. |
Stewart, J. P.,
E. J. Usherwood,
A. Ross,
H. Dyson, and T. Nash.
1998.
Lung epithelial cells are a major site of murine gammaherpesvirus persistence.
J. Exp. Med.
187:1941-1951 |
| 70. | Sunil-Chandra, N. P., J. Arno, J. Fazakerley, and A. A. Nash. 1994. Lymphoproliferative disease in mice infected with murine gammaherpesvirus 68. Am. J. Pathol. 145:818-826[Abstract]. |
| 71. |
Sunil-Chandra, N. P.,
S. Efstathiou,
J. Arno, and A. A. Nash.
1992.
Virological and pathological features of mice infected with murine gammaherpesvirus 68.
J. Gen. Virol.
73:2347-2356 |
| 72. |
Sunil-Chandra, N. P.,
S. Efstathiou, and A. A. Nash.
1992.
Murine gammaherpesvirus 68 establishes a latent infection in mouse B lymphocytes in vivo.
J. Gen. Virol.
73:3275-3279 |
| 73. | Tarodi, B., T. Subramanian, and G. Chinnadurai. 1994. Epstein-Barr virus BHRF1 protein protects against cell death induced by DNA-damaging agents and heterologous viral infection. Virology 201:404-407[Medline]. |
| 74. | Usherwood, E. J., J. P. Stewart, and A. A. Nash. 1996. Characterization of tumor cell lines derived from murine gammaherpesvirus-68-infected mice. J. Virol. 70:6516-6518[Abstract]. |
| 75. |
Usherwood, E. J.,
J. P. Stewart,
K. Robertson,
D. J. Allen, and A. A. Nash.
1996.
Absence of splenic latency in murine gammaherpesvirus 68-infected B cell-deficient mice.
J. Gen. Virol.
77:2819-2825 |
| 75a. | Van Berkel, V., K. Preiter, H. W. Virgin, and S. H. Speck. Identification of a murine gammaherpes virus 68 gene encoding an abundantly secreted protein. Submitted for publication. |
| 75b. | Van Dyk, L. F., J. D. Katz, M. Jacoby, S. H. Speck, and H. W. Virgin. Submitted for publication. |
| 76. | Virgin, H. W., IV, P. Latreille, P. Wamsley, K. Hallsworth, K. E. Weck, A. J. Dal Canto, and S. H. Speck. 1997. Complete sequence and genomic analysis of murine gammaherpesvirus 68. J. Virol. 71:5894-5904[Abstract]. |
| 77. |
Weck, K. E.,
M. L. Barkon,
L. I. Yoo,
S. H. Speck, and H. W. Virgin, IV.
1996.
Mature B cells are required for acute splenic infection, but not for establishment of latency, by murine gammaherpesvirus 68.
J. Virol.
70:6775-6780 |
| 78. | Weck, K. E., A. J. Dal Canto, J. D. Gould, A. K. O'Guin, K. A. Roth, J. E. Saffitz, S. H. Speck, and H. W. Virgin. 1997. Murine gammaherpesvirus 68 causes large vessel arteritis in mice lacking interferon-gamma responsiveness: a new model for virus induced vascular disease. Nat. Med. 3:1346-1353[Medline]. |
| 78a. | Weck, K. E., S. S. Kim, H. W. Virgin, and S. H. Speck. Macrophages are the major reservoir of latent murine gammaherpesvirus 68 in peritoneal cells. J. Virol., in press. |
| 79. | Zhong, W., and D. Ganem. 1997. Characterization of ribonucleoprotein complexes containing an abundant polyadenylated nuclear RNA encoded by Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8). J. Virol. 71:1207-1212[Abstract]. |
| 80. |
Zhong, W.,
H. Wang,
B. Herndier, and D. Ganem.
1996.
Restricted expression of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) genes in Kaposi's sarcoma.
Proc. Natl. Acad. Sci. USA
93:6641-6646 |
Journal of Virology, March 1999, p. 2321-2332, Vol. 73, No. 3
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Lee, K. S., Groshong, S. D., Cool, C. D., Kleinschmidt-DeMasters, B. K., van Dyk, L. F.
(2009). Murine Gammaherpesvirus 68 Infection of IFN{gamma} Unresponsive Mice: A Small Animal Model for Gammaherpesvirus-Associated B-Cell Lymphoproliferative Disease. Cancer Res.
69: 5481-5489
[Abstract] [Full Text] -
Hwang, S., Wu, T.-T., Tong, L. M., Kim, K. S., Martinez-Guzman, D., Colantonio, A. D., Uittenbogaart, C. H., Sun, R.
(2008). Persistent Gammaherpesvirus Replication and Dynamic Interaction with the Host In Vivo. J. Virol.
82: 12498-12509
[Abstract] [Full Text] -
Li, H., Ikuta, K., Sixbey, J. W., Tibbetts, S. A.
(2008). A Replication-Defective Gammaherpesvirus Efficiently Establishes Long-Term Latency in Macrophages but Not in B Cells In Vivo. J. Virol.
82: 8500-8508
[Abstract] [Full Text] -
DeZalia, M., Speck, S. H.
(2008). Identification of Closely Spaced but Distinct Transcription Initiation Sites for the Murine Gammaherpesvirus 68 Latency-Associated M2 Gene. J. Virol.
82: 7411-7421
[Abstract] [Full Text] -
Forrest, J. C., Speck, S. H.
(2008). Establishment of B-Cell Lines Latently Infected with Reactivation-Competent Murine Gammaherpesvirus 68 Provides Evidence for Viral Alteration of a DNA Damage-Signaling Cascade. J. Virol.
82: 7688-7699
[Abstract] [Full Text] -
Herskowitz, J. H., Siegel, A. M., Jacoby, M. A., Speck, S. H.
(2008). Systematic Mutagenesis of the Murine Gammaherpesvirus 68 M2 Protein Identifies Domains Important for Chronic Infection. J. Virol.
82: 3295-3310
[Abstract] [Full Text] -
Stapler, D., Lee, E. D., Selvaraj, S. A., Evans, A. G., Kean, L. S., Speck, S. H., Larsen, C. P., Gangappa, S.
(2008). Expansion of Effector Memory TCR V{beta}4+CD8+ T Cells Is Associated with Latent Infection-Mediated Resistance to Transplantation Tolerance. J. Immunol.
180: 3190-3200
[Abstract] [Full Text] -
Cush, S. S., Anderson, K. M., Ravneberg, D. H., Weslow-Schmidt, J. L., Flano, E.
(2007). Memory Generation and Maintenance of CD8+ T Cell Function during Viral Persistence. J. Immunol.
179: 141-153
[Abstract] [Full Text] -
Steed, A., Buch, T., Waisman, A., Virgin, H. W. IV
(2007). Gamma Interferon Blocks Gammaherpesvirus Reactivation from Latency in a Cell Type-Specific Manner. J. Virol.
81: 6134-6140
[Abstract] [Full Text] -
Upton, J. W., Speck, S. H.
(2006). Evidence for CDK-Dependent and CDK-Independent Functions of the Murine Gammaherpesvirus 68 v-Cyclin. J. Virol.
80: 11946-11959
[Abstract] [Full Text] -
Allen, R. D. III, Dickerson, S., Speck, S. H.
(2006). Identification of Spliced Gammaherpesvirus 68 LANA and v-Cyclin Transcripts and Analysis of Their Expression In Vivo during Latent Infection. J. Virol.
80: 2055-2062
[Abstract] [Full Text] -
Barton, E. S., Lutzke, M. L., Rochford, R., Virgin, H. W. IV
(2005). Alpha/Beta Interferons Regulate Murine Gammaherpesvirus Latent Gene Expression and Reactivation from Latency. J. Virol.
79: 14149-14160
[Abstract] [Full Text] -
Flano, E., Kayhan, B., Woodland, D. L., Blackman, M. A.
(2005). Infection of Dendritic Cells by a {gamma}2-Herpesvirus Induces Functional Modulation. J. Immunol.
175: 3225-3234
[Abstract] [Full Text] -
Matsumura, S., Fujita, Y., Gomez, E., Tanese, N., Wilson, A. C.
(2005). Activation of the Kaposi's Sarcoma-Associated Herpesvirus Major Latency Locus by the Lytic Switch Protein RTA (ORF50). J. Virol.
79: 8493-8505
[Abstract] [Full Text] -
Flano, E., Jia, Q., Moore, J., Woodland, D. L., Sun, R., Blackman, M. A.
(2005). Early Establishment of {gamma}-Herpesvirus Latency: Implications for Immune Control. J. Immunol.
174: 4972-4978
[Abstract] [Full Text] -
Coleman, H. M., Efstathiou, S., Stevenson, P. G.
(2005). Transcription of the murine gammaherpesvirus 68 ORF73 from promoters in the viral terminal repeats. J. Gen. Virol.
86: 561-574
[Abstract] [Full Text] -
Herskowitz, J., Jacoby, M. A., Speck, S. H.
(2005). The Murine Gammaherpesvirus 68 M2 Gene Is Required for Efficient Reactivation from Latently Infected B Cells. J. Virol.
79: 2261-2273
[Abstract] [Full Text] -
de Lima, B. D., May, J. S., Marques, S., Simas, J. P., Stevenson, P. G.
(2005). Murine gammaherpesvirus 68 bcl-2 homologue contributes to latency establishment in vivo. J. Gen. Virol.
86: 31-40
[Abstract] [Full Text] -
Liang, X., Shin, Y. C., Means, R. E., Jung, J. U.
(2004). Inhibition of Interferon-Mediated Antiviral Activity by Murine Gammaherpesvirus 68 Latency-Associated M2 Protein. J. Virol.
78: 12416-12427
[Abstract] [Full Text] -
Loh, J., Thomas, D. A., Revell, P. A., Ley, T. J., Virgin, H. W. IV
(2004). Granzymes and Caspase 3 Play Important Roles in Control of Gammaherpesvirus Latency. J. Virol.
78: 12519-12528
[Abstract] [Full Text] -
Simas, J. P., Marques, S., Bridgeman, A., Efstathiou, S., Adler, H.
(2004). The M2 gene product of murine gammaherpesvirus 68 is required for efficient colonization of splenic follicles but is not necessary for expansion of latently infected germinal centre B cells. J. Gen. Virol.
85: 2789-2797
[Abstract] [Full Text] -
Moorman, N. J., Lin, C. Y., Speck, S. H.
(2004). Identification of Candidate Gammaherpesvirus 68 Genes Required for Virus Replication by Signature-Tagged Transposon Mutagenesis. J. Virol.
78: 10282-10290
[Abstract] [Full Text] -
Deng, H., Chu, J. T., Park, N.-H., Sun, R.
(2004). Identification of cis Sequences Required for Lytic DNA Replication and Packaging of Murine Gammaherpesvirus 68. J. Virol.
78: 9123-9131
[Abstract] [Full Text] -
Obar, J. J., Crist, S. G., Leung, E. K., Usherwood, E. J.
(2004). IL-15-Independent Proliferative Renewal of Memory CD8+ T Cells in Latent Gammaherpesvirus Infection. J. Immunol.
173: 2705-2714
[Abstract] [Full Text] -
Flano, E., Hardy, C. L., Kim, I.-J., Frankling, C., Coppola, M. A., Nguyen, P., Woodland, D. L., Blackman, M. A.
(2004). T Cell Reactivity during Infectious Mononucleosis and Persistent Gammaherpesvirus Infection in Mice. J. Immunol.
172: 3078-3085
[Abstract] [Full Text] -
Townsley, A. C., Dutia, B. M., Nash, A. A.
(2004). The M4 Gene of Murine Gammaherpesvirus Modulates Productive and Latent Infection In Vivo. J. Virol.
78: 758-767
[Abstract] [Full Text] -
Moorman, N. J., Willer, D. O., Speck, S. H.
(2003). The Gammaherpesvirus 68 Latency-Associated Nuclear Antigen Homolog Is Critical for the Establishment of Splenic Latency. J. Virol.
77: 10295-10303
[Abstract] [Full Text] -
Martinez-Guzman, D., Rickabaugh, T., Wu, T.-T., Brown, H., Cole, S., Song, M. J., Tong, L., Sun, R.
(2003). Transcription Program of Murine Gammaherpesvirus 68. J. Virol.
77: 10488-10503
[Abstract] [Full Text] -
Macrae, A. I., Usherwood, E. J., Husain, S. M., Flano, E., Kim, I.-J., Woodland, D. L., Nash, A. A., Blackman, M. A., Sample, J. T., Stewart, J. P.
(2003). Murid Herpesvirus 4 Strain 68 M2 Protein Is a B-Cell-Associated Antigen Important for Latency but Not Lymphocytosis. J. Virol.
77: 9700-9709
[Abstract] [Full Text] -
Marques, S., Efstathiou, S., Smith, K. G., Haury, M., Simas, J. P.
(2003). Selective Gene Expression of Latent Murine Gammaherpesvirus 68 in B Lymphocytes. J. Virol.
77: 7308-7318
[Abstract] [Full Text] -
Pavlova, I. V., Virgin, H. W. IV, Speck, S. H.
(2003). Disruption of Gammaherpesvirus 68 Gene 50 Demonstrates that Rta Is Essential for Virus Replication. J. Virol.
77: 5731-5739
[Abstract] [Full Text] -
Flano, E., Kim, I.-J., Moore, J., Woodland, D. L., Blackman, M. A.
(2003). Differential {gamma}-Herpesvirus Distribution in Distinct Anatomical Locations and Cell Subsets During Persistent Infection in Mice. J. Immunol.
170: 3828-3834
[Abstract] [Full Text] -
Jia, Q., Sun, R.
(2003). Inhibition of Gammaherpesvirus Replication by RNA Interference. J. Virol.
77: 3301-3306
[Abstract] [Full Text] -
Ebrahimi, B., Dutia, B. M., Roberts, K. L., Garcia-Ramirez, J. J., Dickinson, P., Stewart, J. P., Ghazal, P., Roy, D. J., Nash, A. A.
(2003). Transcriptome profile of murine gammaherpesvirus-68 lytic infection. J. Gen. Virol.
84: 99-109
[Abstract] [Full Text] -
Flano, E., Kim, I.-J., Woodland, D. L., Blackman, M. A.
(2002). {gamma}-Herpesvirus Latency Is Preferentially Maintained in Splenic Germinal Center and Memory B Cells. JEM
196: 1363-1372
[Abstract] [Full Text] -
Gangappa, S., Kapadia, S. B., Speck, S. H., Virgin, H. W. IV
(2002). Antibody to a Lytic Cycle Viral Protein Decreases Gammaherpesvirus Latency in B-Cell-Deficient Mice. J. Virol.
76: 11460-11468
[Abstract] [Full Text] -
Seet, B. T., McFadden, G.
(2002). Viral chemokine-binding proteins. J. Leukoc. Biol.
72: 24-34
[Abstract] [Full Text] -
Clambey, E. T., Virgin, H. W. IV, Speck, S. H.
(2002). Characterization of a Spontaneous 9.5-Kilobase-Deletion Mutant of Murine Gammaherpesvirus 68 Reveals Tissue-Specific Genetic Requirements for Latency. J. Virol.
76: 6532-6544
[Abstract] [Full Text] -
Ahn, J. W., Powell, K. L., Kellam, P., Alber, D. G.
(2002). Gammaherpesvirus Lytic Gene Expression as Characterized by DNA Array. J. Virol.
76: 6244-6256
[Abstract] [Full Text] -
Coulter, L. J., Reid, H. W.
(2002). Isolation and expression of three open reading frames from ovine herpesvirus-2. J. Gen. Virol.
83: 533-543
[Abstract] [Full Text] -
Jacoby, M. A., Virgin, H. W. IV, Speck, S. H.
(2002). Disruption of the M2 Gene of Murine Gammaherpesvirus 68 Alters Splenic Latency following Intranasal, but Not Intraperitoneal, Inoculation. J. Virol.
76: 1790-1801
[Abstract] [Full Text] -
Bridgeman, A., Stevenson, P. G., Simas, J. P., Efstathiou, S.
(2001). A Secreted Chemokine Binding Protein Encoded by Murine Gammaherpesvirus-68 Is Necessary for the Establishment of a Normal Latent Load. JEM
194: 301-312
[Abstract] [Full Text] -
Rochford, R., Lutzke, M. L., Alfinito, R. S., Clavo, A., Cardin, R. D.
(2001). Kinetics of Murine Gammaherpesvirus 68 Gene Expression following Infection of Murine Cells in Culture and in Mice. J. Virol.
75: 4955-4963
[Abstract] [Full Text] -
Macrae, A. I., Dutia, B. M., Milligan, S., Brownstein, D. G., Allen, D. J., Mistrikova, J., Davison, A. J., Nash, A. A., Stewart, J. P.
(2001). Analysis of a Novel Strain of Murine Gammaherpesvirus Reveals a Genomic Locus Important for Acute Pathogenesis. J. Virol.
75: 5315-5327
[Abstract] [Full Text] -
Wakeling, M. N., Roy, D. J., Nash, A. A., Stewart, J. P.
(2001). Characterization of the murine gammaherpesvirus 68 ORF74 product: a novel oncogenic G protein-coupled receptor. J. Gen. Virol.
82: 1187-1197
[Abstract] [Full Text] -
Nicholas, J
(2000). Evolutionary aspects of oncogenic herpesviruses. Mol. Pathol.
53: 222-237
[Abstract] [Full Text] -
Usherwood, E. J., Roy, D. J., Ward, K., Surman, S. L., Dutia, B. M., Blackman, M. A., Stewart, J. P., Woodland, D. L.
(2000). Control of Gammaherpesvirus Latency by Latent Antigen-specific CD8+ T Cells. JEM
192: 943-952
[Abstract] [Full Text] -
Hall, K. T., Giles, M. S., Goodwin, D. J., Calderwood, M. A., Carr, I. M., Stevenson, A. J., Markham, A. F., Whitehouse, A.
(2000). Analysis of Gene Expression in a Human Cell Line Stably Transduced with Herpesvirus Saimiri. J. Virol.
74: 7331-7337
[Abstract] [Full Text] -
van Dyk, L. F., Virgin, H. W. IV, Speck, S. H.
(2000). The Murine Gammaherpesvirus 68 v-Cyclin Is a Critical Regulator of Reactivation from Latency. J. Virol.
74: 7451-7461
[Abstract] [Full Text] -
van Berkel, V., Barrett, J., Tiffany, H. L., Fremont, D. H., Murphy, P. M., McFadden, G., Speck, S. H., Virgin, H. W. IV
(2000). Identification of a Gammaherpesvirus Selective Chemokine Binding Protein That Inhibits Chemokine Action. J. Virol.
74: 6741-6747
[Abstract] [Full Text] -
Hoge, A. T., Hendrickson, S. B., Burns, W. H.
(2000). Murine Gammaherpesvirus 68 Cyclin D Homologue Is Required for Efficient Reactivation from Latency. J. Virol.
74: 7016-7023
[Abstract] [Full Text] -
Flano, E., Husain, S. M., Sample, J. T., Woodland, D. L., Blackman, M. A.
(2000). Latent Murine {gamma}-Herpesvirus Infection Is Established in Activated B Cells, Dendritic Cells, and Macrophages. J. Immunol.
165: 1074-1081
[Abstract] [Full Text] -
Bellows, D. S., Chau, B. N., Lee, P., Lazebnik, Y., Burns, W. H., Hardwick, J. M.
(2000). Antiapoptotic Herpesvirus Bcl-2 Homologs Escape Caspase-Mediated Conversion to Proapoptotic Proteins. J. Virol.
74: 5024-5031
[Abstract] [Full Text] -
Clambey, E. T., Virgin, H. W. IV, Speck, S. H.
(2000). Disruption of the Murine Gammaherpesvirus 68 M1 Open Reading Frame Leads to Enhanced Reactivation from Latency. J. Virol.
74: 1973-1984
[Abstract] [Full Text] -
Parry, C. M., Simas, J. P., Smith, V. P., Stewart, C. A., Minson, A. C., Efstathiou, S., Alcami, A.
(2000). A Broad Spectrum Secreted Chemokine Binding Protein Encoded by a Herpesvirus. JEM
191: 573-578
[Abstract] [Full Text] -
Peacock, J. W., Bost, K. L.
(2000). Infection of intestinal epithelial cells and development of systemic disease following gastric instillation of murine gammaherpesvirus-68. J. Gen. Virol.
81: 421-429
[Abstract] [Full Text] -
Brooks, J. W., Hamilton-Easton, A. M., Christensen, J. P., Cardin, R. D., Hardy, C. L., Doherty, P. C.
(1999). Requirement for CD40 Ligand, CD4+ T Cells, and B Cells in an Infectious Mononucleosis-Like Syndrome. J. Virol.
73: 9650-9654
[Abstract] [Full Text] -
Kapadia, S. B., Molina, H., van Berkel, V., Speck, S. H., Virgin, H. W. IV
(1999). Murine Gammaherpesvirus 68 Encodes a Functional Regulator of Complement Activation. J. Virol.
73: 7658-7670
[Abstract] [Full Text] -
Stevenson, P. G., Belz, G. T., Castrucci, M. R., Altman, J. D., Doherty, P. C.
(1999). A gamma -herpesvirus sneaks through a CD8+ T cell response primed to a lytic-phase epitope. Proc. Natl. Acad. Sci. USA
96: 9281-9286
[Abstract] [Full Text] -
Coppola, M. A., Flano, E., Nguyen, P., Hardy, C. L., Cardin, R. D., Shastri, N., Woodland, D. L., Blackman, M. A.
(1999). Apparent MHC-Independent Stimulation of CD8+ T Cells In Vivo During Latent Murine Gammaherpesvirus Infection. J. Immunol.
163: 1481-1489
[Abstract] [Full Text] -
Husain, S. M., Usherwood, E. J., Dyson, H., Coleclough, C., Coppola, M. A., Woodland, D. L., Blackman, M. A., Stewart, J. P., Sample, J. T.
(1999). Murine gammaherpesvirus M2 gene is latency-associated and its protein a target for CD8+ T lymphocytes. Proc. Natl. Acad. Sci. USA
96: 7508-7513
[Abstract] [Full Text] -
Weck, K. E., Kim, S. S., Virgin, H. W. IV, Speck, S. H.
(1999). B Cells Regulate Murine Gammaherpesvirus 68 Latency. J. Virol.
73: 4651-4661
[Abstract] [Full Text] -
van Dyk, L. F., Hess, J. L., Katz, J. D., Jacoby, M., Speck, S. H., Virgin, H. W. IV
(1999). The Murine Gammaherpesvirus 68 v-Cyclin Gene Is an Oncogene That Promotes Cell Cycle Progression in Primary Lymphocytes. J. Virol.
73: 5110-5122
[Abstract] [Full Text] -
van Berkel, V., Preiter, K., Virgin, H. W. IV, Speck, S. H.
(1999). Identification and Initial Characterization of the Murine Gammaherpesvirus 68 Gene M3, Encoding an Abundantly Secreted Protein. J. Virol.
73: 4524-4529
[Abstract] [Full Text] -
Gangappa, S., van Dyk, L. F., Jewett, T. J., Speck, S. H., Virgin, H. W. IV
(2002). Identification of the In Vivo Role of a Viral bcl-2. JEM
195: 931-940
[Abstract] [Full Text] -
Blackman, M. A., Flano, E.
(2002). Persistent {gamma}-herpesvirus Infections: What Can We Learn from an Experimental Mouse Model?. JEM
195: F29-F32
[Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»