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Journal of Virology, August 2000, p. 7451-7461, Vol. 74, No. 16
Department of Pathology and Immunology and
Department of Molecular Microbiology, Washington University School
of Medicine, St. Louis, Missouri
Received 12 April 2000/Accepted 25 May 2000
Gamma-2 herpesviruses encode a homolog of mammalian D-type cyclins.
The v-cyclin encoded by murine gammaherpesvirus 68 ( Murine gammaherpesvirus 68 ( Manipulation of the host cell cycle is a shared feature of
gammaherpesviruses, and one focus for this regulation is the
D-type cyclins. EBV infection (or expression of the EBV
latency-associated membrane protein 1) upregulates expression of host
cyclin D2 (3, 9, 63). In contrast, the gamma-2 herpesviruses
HVS (32, 47), KSHV (2, 47, 52), and The biochemistry of the KSHV and HVS proteins has been intensively
studied. The KSHV and HVS v-cyclins, in conjunction with cdk's, have
been demonstrated to phosphorylate the retinoblastoma protein (pRb) and
promote cell cycle progression, like their mammalian homologs (2,
8). The KSHV and HVS v-cyclins predominantly bind cdk6.
Overexpression of the KSHV v-cyclin in cells expressing high levels of
cdk6 results in apoptosis (48), suggesting that there may be
other viral genes responsible for preventing apoptosis (e.g., v-bcl2).
Several unusual properties of the v-cyclins have also been noted. The
v-cyclin ORFs lack an LXCXE motif present in mammalian cyclin D
proteins and thought to be important in direct binding to pRb (20,
25). The HVS and KSHV v-cyclins are able to bind multiple cdk's,
rather than being restricted to binding cdk4 and cdk6 (27, 32,
39), and have a broadened substrate range, being capable of
phosphorylating both pRb and histone H1 in vitro. In addition, the HVS
and KSHV v-cyclin-cdk complexes are resistant to regulation by the
cellular cyclin/cdk inhibitors of both the INK4 and Kip1 families
(24, 41, 65). The resistance to inhibitors impacts not only
the v-cyclin-cdk complex itself but also the activity of other
cyclin-cdk's by means of decreased availability and
redistribution of the inhibitors (42). The KSHV
v-cyclin is resistant to inhibition by both p21 and p27 but is only
known to phosphorylate p27, which is then actively degraded (24,
41). The HVS v-cyclin structure has been determined (56,
60), and although this analysis revealed conservation of
mammalian cyclin structure in regions critical for interaction with
cdk's, differences in other regions of the v-cyclin structure may
explain some of the noted functional differences.
We have previously shown that the Viruses and tissue culture.
Generation of mutant
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The Murine Gammaherpesvirus 68 v-Cyclin Is a
Critical Regulator of Reactivation from Latency
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
HV68) induces
cell cycle progression and is an oncogene (L. F. van Dyk, J. L. Hess, J. D. Katz, M. Jacoby, S. H. Speck, and H. W. Virgin IV, J. Virol. 73:5110-5122, 1999). However, the role of
the pro-proliferative v-cyclins in gamma-2 herpesvirus pathogenesis is
not known. Here we report the generation and characterization of a
HV68 v-cyclin mutant (v-cyclin.LacZ) that is unable to
express a functional v-cyclin protein. Notably, although the HV68
v-cyclin is expressed from an early-late lytic transcript,
v-cyclin.LacZ replicated normally in fibroblasts in vitro and
during acute infection in the spleen, liver, and lungs in vivo.
Moreover, v-cyclin.LacZ exhibited wild-type (wt) virulence
in mice with severe combined immunodeficiency. In addition,
in a model of HV68-induced chronic disease in mice lacking the gamma
interferon receptor (IFNR
/),
v-cyclin.LacZ virus was similar to wt HV68 in terms of
the incidence of mortality and vasculitis. Further analysis revealed that the frequencies of splenocytes and peritoneal cells harboring the
latent HV68 genome in normal and B-cell-deficient mice infected with
wt HV68 or v-cyclin.LacZ were very similar. However,
v-cyclin.LacZ was significantly compromised in its capacity to
reactivate from latency. This phenotype was conclusively mapped to the
v-cyclin gene by (i) generating a marker rescue virus
(v-cyclin.MR) from the v-cyclin.LacZ mutant, which
restored the frequency of cells in which virus reactivated from latency
to the levels observed with wt HV68; and (ii) generating a second
v-cyclin mutant virus containing a translation stop codon within the
v-cyclin gene (v-cyclin.stop), which was compromised in reactivation
from latency. These studies demonstrate that despite expression as a
lytic cycle gene, the pro-proliferative HV68 v-cyclin is not
required for HV68 replication either in vitro or during acute
infection in vivo but rather is a critical determinant of
reactivation from latency.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
HV68;
also referred to as MHV68) infection of mice is a developing
small-animal model for analysis of gammaherpesvirus pathogenesis
(reviewed in references 18, 19, 45, 46, 64, and
69). HV68 is a gamma-2 herpesvirus which is
closely related to the human and primate gammaherpesviruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and herpesvirus saimiri (HVS) (22, 67). Acute
HV68 infection of laboratory mice is cleared by 2 to 3 weeks
postinfection, with the concomitant establishment of a presumably
life-long latent infection. Notably, several HV68 genes that are
also present in HVS, KSHV, and/or EBV have been identified as candidate
latent genes by transcriptional analysis of latently infected mice
(68). These genes include those encoding the viral bcl-2
(v-bcl2) homolog and the viral G-protein-coupled receptor (v-GPCR)
homolog as well as gene 73, which in KSHV encodes the
latency-associated nuclear antigen (LANA) (11, 35,
50). In addition, KSHV, HVS, and HV68 have in common a
complement-regulatory protein homolog (1, 2, 26, 33, 67),
and in both HVS and HV68 this protein occurs in membrane-bound and
soluble isoforms (26, 33). These studies argue strongly that
HV68 shares mechanisms of pathogenesis with other gammaherpesviruses.
HV68
(67) all contain open reading frames (ORFs) predicted to
encode homologs of mammalian D-type cyclins. The viral cyclins
(v-cyclins) exhibit 25 to 31% identity to mammalian D-type cyclins and
26 to 32% identity to each other, and they are positionally
conserved in the gamma-2 herpesvirus genomes (67).
Notably, the highest level of sequence conservation among these
homologs is within the cyclin box, a domain demonstrated to be
essential for cyclin-dependent kinase (cdk) binding (29, 31, 37,
38, 43, 51).
HV68 v-cyclin promotes cell cycle
progression in primary lymphocytes and can function as an oncogene in
transgenic mice (66). Thus, the known gamma-2 herpesvirus
v-cyclins have all been shown to promote cell cycle progression
(66). While much is known about the functional properties of
the v-cyclins, their role in the pathogenesis of gamma-2 herpesviruses has not been defined. Here we present a detailed analysis of acute and
latent infection in mice, using a recombinant HV68 mutant lacking a
functional v-cyclin gene.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
HV68 clone WUMS (ATCC VR1465)
was passaged and grown, and the titer was determined as previously
described (13, 70, 71). NIH 3T12 cells and murine embryonic
fibroblasts (MEFs) were grown in Dulbecco's modified Eagle medium
(DMEM) supplemented with 10% fetal calf serum, 100 U of penicillin/ml,
100 mg of streptomycin/ml, and 2 mM L-glutamine. MEFs were
obtained from BALB/c or C57BL/6 mouse embryos as described previously
(70).
HV68 viruses.
All recombinant
viruses were generated by homologous recombination following
cotransfection of NIH 3T12 cells with viral DNA and gene-targeting
plasmid DNA as previously described (13). The parental
genomic subclone for the targeting plasmids was constructed by
insertion of the 3,723-bp BamHI-BsrGI fragment of
HV68 (bp 101654 to 105377; WUMS sequence [67]),
containing gene 72 and flanking sequence, into the corresponding sites
of Litmus 38 (New England Biolabs, Beverley, Mass.) to generate a
plasmid called pL3700. The mutant targeting vector (pORF72-LacZ) was
generated by inserting a 
-galactosidase expression cassette driven
by the human cytomegalovirus (HCMV) immediate-early promoter-enhancer (HindIII-XmaI fragment from
pHCMV-LacZ-MP-1; a kind gift of Paul Olivo [unpublished]) into
pL3700. The plasmid pORF72-LacZ contains the LacZ expression cassette
in place of 475 bp of gene 72 (v-cyclin), beginning with the ATG
defining the translation start site of the ORF (from the
NcoI site at bp 103179 to the NsiI site at bp 102704). The plasmid pL3700-stop was generated by insertion of a 16-bp
stop linker containing a unique HpaI site (15) at
the PmlI site (bp 103021). All targeting plasmids were
sequenced across mutations and cloning junctions and purified on cesium
chloride gradients prior to transfection (54).
HV68 genomic DNA (2 µg) and pORF72-LacZ plasmid linearized by digestion with
NheI (2 µg). Virus was harvested 5 to 8 days
posttransfection, aliquoted, and stored at 
80°C. Recombinant virus
was then identified by blue plaque morphology and purified as described
previously (13). Briefly, recombinant viruses were
identified as individual blue plaques after X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactoside) staining of
infected NIH 3T12 monolayers and isolated from methyl cellulose-overlaid monolayers (49).
80°C. Titers of all
viruses were determined by plaque assay at least three independent times.
Southern blotting. Viral DNA for Southern blot analysis and for transfection was generated by infection of NIH 3T12 cells at an MOI of 0.5. Infected-cell cultures were harvested at 50% CPE, between days 4 and 6 postinfection, and DNA was prepared as previously described (67). One microgram of viral DNA was digested with either EcoRI, BamHI, or NsiI plus NotI. EcoRI and BamHI digests result in diagnostic fragments for the v-cyclin region, and the NsiI-NotI digestion is diagnostic for deletions at the left end of the viral genome (13). Digests were electrophoresed on 1% agarose gels and transferred by alkaline transfer to Nytran nylon membranes (Turboblot; Schleicher & Schuell, Keene, N.H.) according to the manufacturer's recommendations. Southern blots were probed with the 3,723-bp BamHI-BsrGI fragment of pL3700 for analysis of the v-cyclin region or with a 2,926-bp HindIII-EcoRV fragment of the HindIII G plasmid containing sequences from gene 6 (encoding a single-stranded DNA-binding protein) for evaluation of the left end of the viral genome (23). The probe was 32P labeled by random primer extension according to the manufacturer's recommendations (Megaprime DNA labeling kit; Amersham International). Blots were hybridized at 68°C and washed with two changes of 2× SSC-0.1% sodium dodecyl sulfate (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) followed by two changes of 0.5× SSC-0.1% sodium dodecyl sulfate prior to exposure to film (54).
Immunoblotting.
Polyclonal rabbit antiserum (Cocalico,
Reamstown, Pa.) was generated by using purified bacterially expressed
v-cyclin protein as described previously (66). Whole-cell
lysates for use in immunoblot analyses were made by resuspending frozen
infected-cell pellets in 1× reducing loading sample buffer and boiling
for 10 min. A total of 2.5 × 105 cell equivalents was
loaded per lane on 15% polyacrylamide gels and transferred to a Hybond
N membrane (Amersham). Blots were blocked for 45 min in
phosphate-buffered saline containing 5% nonfat milk, 5% normal goat
serum, and 0.05% Tween 20 and then incubated with polyclonal rabbit
anti-v-cyclin serum (1:2,000 dilution) overnight at room temperature.
Blots were washed three times in phosphate-buffered saline containing
0.05% Tween 20, incubated in horseradish peroxidase-conjugated donkey
anti-rabbit antiserum (1:5,000 dilution; Jackson Immunoresearch),
washed three times with PBS containing 0.05% Tween 20, and developed
with the ECL chemiluminescence reagent (Amersham). A monoclonal
antibody against -actin (1:1,000 dilution; Sigma catalog no. AC74),
used to monitor loading of cell extracts, was detected with horseradish peroxidase-conjugated donkey anti-mouse antiserum (1:5,000 dilution; Jackson Immunoresearch).
Plaque assays and determination of viral titers. Plaque assays were performed on NIH 3T12 cells as previously described (13). Briefly, organs for which virus titers were to be determined were thawed and homogenized with a Ten Broek tissue grinder prior to dilution and plating onto NIH 3T12 cells. Infection was performed at 37°C for 1 h before cells were overlaid with medium containing Noble agar. All titers were determined in parallel with a laboratory standard stock of known titer. The limit of detection of the assay used is 50 PFU per organ.
In vitro growth.
Viral growth in vitro was determined by
infection of NIH 3T12 cells at an MOI of 5 PFU per cell, with removal
of the inoculum after 1 h of infection to measure a single cycle
of virus replication, or at 0.05 PFU per cell to measure multiple
cycles of virus replication. Cells and supernatants were collected at
various times postinfection and frozen at 80°C. Samples were then
subjected to four cycles of freezing and thawing, and virus was then
quantitated by plaque assay (sensitivity, 50 PFU). Growth of wild-type
(wt) HV68 in contact-inhibited MEFs was compared with that of
v-cyclin.LacZ by infecting MEF monolayers with 1 PFU per well
in 96-well plates. Four independent sets of dilutions of each virus
were plated in 24 wells of 96-well plates and scored for appearance of
CPE. Six wells from each dilution were harvested for determination of
virus titers by plaque assay.
Mice, infections, and organ harvests.
Gamma interferon
receptor-deficient (IFNR/) mice on a 129 background
were obtained from Michel Aguet (44). B-cell-deficient mice
(B6µMT; C57BL/6J-Igh-6tm1Cgn) (36) were
obtained from Jackson Laboratories (Bar Harbor, Maine). C.B-17 severe
combined immunodeficient (SCID) mice (5) were obtained from
Emil Unanue and used between 3 to 5 months of age. Mice were bred and
maintained at Washington University School of Medicine in accordance
with all federal and university policies. C57BL/6 mice were purchased
from Jackson Laboratories. Mice were age and sex matched and were used
between 7 to 10 weeks unless otherwise stated. Mice were anesthetized
with metofane prior to infection or sacrifice by cervical dislocation.
Mice were infected by intraperitoneal (i.p.) injection with
106 PFU of virus (unless otherwise stated) in 0.5 ml of
complete DMEM. Upon sacrifice, organs for which virus titers were to be determined were placed in 1 ml of DMEM on ice and frozen at 80°C. Peritoneal exudate cells (PECs) were harvested by peritoneal lavage with 10 ml of DMEM. Virulence in C.B-17 SCID mice was determined by
infection via i.p. injection with medium alone or containing 101, 103, or 106 PFU of virus, and
survival was monitored daily.
R/ mice between 7 to 9 weeks of age were infected i.p. with 2 × 107 PFU of
wt HV68 or v-cyclin.LacZ. At the time of death or sacrifice, the heart and lungs were removed en bloc. The spleen, liver, and kidneys were also harvested. Tissues were fixed in 10%
phosphate-buffered formalin, paraffin embedded, serial sectioned, and
stained with hematoxylin and eosin. Slides were independently read by
L.V.D., H.W.V., and S. Kapadia, who were blinded to their identities. Aortic lesions were scored as follows: 0, normal artery; 1, mild intimal and adventitial thickening with minimal infiltrates; 2, pronounced intimal and adventitial mononuclear infiltrates but no
neutrophilic infiltrates; 3, pronounced intimal and adventitial mononuclear infiltrates and neutrophilic infiltrates at the adventitial and medial borders; 4, pronounced intimal and adventitial mononuclear infiltrates and presence of neutrophilic infiltrates in the media; and
5, severe lesions with extensive neutrophilic infiltrates and/or
necrosis in the media (A. J. Dal Canto, H. W. Virgin IV, and
S. H. Speck, submitted for publication). Samples from the same
mice were examined for the presence of fibrosis or atrophy of the
spleen as previously described (21).
Quantitation of cells harboring the HV68 genome.
The
frequency of cells harboring the HV68 genome was determined by a
limiting-dilution nested-PCR assay that amplifies HV68 gene 50 sequences with approximately single-copy sensitivity, as described
previously (72, 73). Briefly, PECs and splenocytes were
harvested from latently infected mice and frozen in 10% dimethyl sulfoxide at 80°C. Cells were thawed, counted, and resuspended in
an isotonic buffer. A limiting-dilution series of cells harvested from
latently infected mice was then generated and plated in 96-well PCR
plates (Costar). Uninfected NIH 3T12 cells were added to provide a
constant cell number of 104 cells per well. Cells were then
lysed with proteinase K at 56°C for 6 h, and the lysate was
heated inactivated at 95°C for 15 min prior to PCR amplification. One
microliter of the 20-µl first-round PCR product served as a template
for the second round of PCR amplification. Products were analyzed by
ethidium bromide staining of a 1.5% agarose gel. Ten PCRs were
analyzed for each cell dilution, with six dilutions per sample per
experiment. Control reactions of uninfected cells (negative control;
six reactions/plate) or plasmid DNA (pBamHIN) of known copy number
(positive control; six reactions/plate each of 10, 1, and 0.1 copy of
plasmid DNA) were included in each experiment. There were no
false-positive PCRs in any analyses reported here, and all assays
demonstrated approximately a one-copy sensitivity for plasmid DNA.
Ex vivo reactivation from latency. The frequency of cells capable of reactivating virus from latency was determined by a limiting-dilution ex vivo reactivation assay, as previously described (70, 73). Briefly, PECs and splenocytes were harvested from infected mice between days 42 and 45 postinfection, and single-cell suspensions were generated. Cells were resuspended in complete DMEM and plated in a twofold-dilution series (starting at 105 splenocytes per well and at 8 × 104 to 16 × 104 PECs per well) onto MEF monolayers in 96-well tissue culture plates. Wells were scored microscopically for CPE at 21 days postplating. Some wells were replated onto fresh MEF monolayers for final determination of infectious-virus titers, particularly wells containing large numbers of cells for which viral CPE was difficult to discern. Twenty-four wells were plated per dilution, and 12 dilutions were plated per experimental sample. Preformed infectious virus was detected by plating parallel cell samples, which had been subjected to mechanical disruption, onto MEF monolayers. Mechanically disrupted samples contained <1% live cells, and thus the presence of preformed virus, rather than viral reactivation from latently infected cells, could be detected (70, 72, 73).
Statistical methods. All data was analyzed by using GraphPad Prism software (GraphPad Software, San Diego, Calif.). Survival data were plotted and statistically analyzed with the Mantel-Haenszel test; to correct for the number of postexperimental comparisons in a conservative way, we multiplied calculated P values by the total number of comparisons between survival curves (significant P values are indicated in the relevant figure legends). Titer data were statistically analyzed with the nonparametric Mann-Whitney test. Severity of aortitis was scored by three independent blinded readers and analyzed with the nonparametric Mann-Whitney test. The frequencies of reactivation and genome-positive cells were statistically analyzed by paired t test. Frequencies of reactivation and viral-genome-positive cells were obtained from the cell number at which 63% of the wells scored positive for either reactivating virus or the presence of the viral genome based on the Poisson distribution; data were subjected to nonlinear-regression analysis to obtain the single-cell frequency for each limiting-dilution analysis.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Targeted disruption of the HV68 v-cyclin gene.
The
v-cyclin-deficient HV68 v-cyclin.LacZ was generated by
insertion of a LacZ expression cassette, containing the
-galactosidase gene under the control of the HCMV immediate-early
promoter-enhancer (see Materials and Methods). The LacZ expression
cassette insertion replaced 475 bp of HV68 sequence, from bp 103179 to 102704 (WUMS sequence [67]), beginning at the
predicted translation initiation codon (Fig.
1A). This
deletion-insertion does not disrupt any other known viral coding
sequences. v-cyclin.LacZ was identified by blue plaque
morphology and plaque purified to homogeneity. The structure of
v-cyclin.LacZ was confirmed by Southern blot analyses of
EcoRI- and BamHI-digested viral DNA probed with a 32P-labeled fragment spanning the region of the viral
genome containing the v-cyclin gene (Fig. 1A and B) (the probe
contained viral sequences from bp 101654 to 105377). As expected, the
v-cyclin region probe hybridized to 3.7- and 1.6-kb BamHI
fragments with v-cyclin.LacZ, compared to a 5.2-kb
BamHI fragment with wt HV68, and to 10.8-, 5.2-, and
0.5-kb EcoRI fragments with v-cyclin.LacZ virus,
compared to 10.8-, 5.2-, and 0.9-kb fragments with wt HV68 (Fig.
1B).
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HV68 genome during the generation of mutant viruses
(13), additional Southern blot analyses were performed to
confirm the integrity of the left end of the viral genome (Fig. 1C).
Viral DNA was digested with NotI and NsiI (Fig.
1A) and probed with a fragment of the HV68 genome containing ORF6
(bp 11100 to 14026). The hybridization of the probe to a 14.8-kb
fragment with wt and mutant viruses (Fig. 1C), and the lack of smaller fragments corresponding to deletions, demonstrated the integrity of the
left end of the viral genome.
We constructed a marker rescue virus (v-cyclin.MR)
in which a targeting vector containing wt v-cyclin sequences was
used to regenerate wt virus from the v-cyclin.LacZ mutant (Fig.
1A). v-cyclin.MR was identified by white plaque selection and
plaque purified to homogeneity. Southern blot analysis of
v-cyclin.MR (Fig. 1B and C) demonstrated that its hybridization
pattern was identical to that of wt HV68. When experiments
demonstrated a phenotypic difference between wt HV68 and
v-cyclin.LacZ, we repeated them with the v-cyclin.MR
virus, thereby excluding the possible contribution of spurious
mutations at distal sites to observed phenotypic differences (see below).
To rule out the possibility that the LacZ expression cassette
contributed to phenotypic changes (e.g., by altering expression of
adjacent genes), as we have previously shown can occur with some
HV68 mutants (13), we replaced the LacZ expression
cassette with a v-cyclin gene containing a translation stop codon at
the PmlI site in the v-cyclin ORF (Fig. 1A, v-cyclin .stop).
This introduced a translation stop codon at the beginning of the highly conserved cyclin box and thus is predicted to encode a nonfunctional 53-amino-acid truncated v-cyclin protein. Southern blot analyses were
performed to confirm the genotype of the v-cyclin.stop virus (Fig. 1B
and C).
Equal numbers of NIH 3T12 cells infected with wt HV68,
v-cyclin.LacZ, v-cyclin.MR, or v-cyclin.stop and
mock-infected cells were analyzed by immunoblotting with a rabbit
polyclonal antiserum generated against bacterially expressed v-cyclin
(66). The v-cyclin protein was readily detected in extracts
prepared from cells infected with wt HV68 or v-cyclin.MR
virus, but not in those of v-cyclin.LacZ-, v-cyclin.stop-, or
mock-infected cells (Fig. 1D). Subsequent incubation of the immunoblot
with a monoclonal antibody against -actin demonstrated equal loading
of cellular extract in all lanes (Fig. 1D).
HV68 v-cyclin.LacZ replicates normally in immortalized
and primary mouse fibroblasts in vitro.
We have previously shown
that the v-cyclin transcript and protein are abundantly expressed
during virus replication in murine fibroblasts (66) and may
therefore be important for lytic-virus replication. We therefore
compared the growth of wt HV68 to that of v-cyclin.LacZ in a single
round of replication and in multiple rounds of replication in NIH 3T12
fibroblasts. In both single and multiple cycles of replication,
v-cyclin.LacZ replication was indistinguishable from that of wt
HV68 (Fig. 2A and B), demonstrating that v-cyclin is not required for efficient replication in an immortalized murine cell line.
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0.0001).
MEFs were infected with one PFU of wt or v-cyclin.LacZ virus
per well of 96-well plates (~10,000 cells/well), and CPE was
monitored over the course of 15 days. There was no difference between
growth of wt HV68 and growth of v-cyclin.LacZ as determined by measuring the induction of CPE (Fig. 2C). To quantitate the amount
of virus produced, six wells each of wt HV68- or
v-cyclin.LacZ-infected cells were harvested at days 6, 8, and
14. The amount of virus produced by infection of MEFs with wt HV68
was equivalent, by plaque assay, to that produced by infection with
v-cyclin.LacZ (Fig. 2D). Thus, the v-cyclin is not essential
for virus replication in either immortal or primary mouse fibroblasts.
The HV68 v-cyclin is dispensable for acute infection in vivo,
virulence in SCID mice, and induction of chronic pathology in
IFNR/ mice.
We assessed the impact of knocking
out the v-cyclin gene on acute and persistent HV68 infection by
comparing wt and mutant viruses in three experimental settings known to
involve virus replication in tissues: (i) acute virus replication at
early times after i.p. virus inoculation; (ii) kinetics of lethality in
C.B-17 SCID mice (5); and (iii) induction of elastic
arteritis and mortality in IFNR/ mice
(71). Acute virus replication in vivo was assessed by determining viral titers in the spleens, livers, and lungs of C57BL/6
(B6) mice 4 or 9 days after infection with 106 PFU of wt
HV68 or v-cyclin.LacZ. Consistent with the in vitro replication data, there were no significant differences in acute virus
replication in these organs (Fig. 3A). As
an independent measure of virus replication in vivo, the virulence of
wt HV68 was compared to that of v-cyclin.LacZ by monitoring
the kinetics of lethality in SCID mice. SCID mice were infected with
101, 103, or 106 PFU of wt HV68
or v-cyclin.LacZ, and survival was monitored (Fig. 3B). At each
dose, wt HV68 and v-cyclin.LacZ had very similar 50%
survival times, and, as expected, higher doses resulted in earlier
lethality. There were some differences in the survival times of a few
infected SCID mice, but no consistent trend was observed (Fig. 3B). A
few wt HV68-infected mice survived longer than any
v-cyclin.LacZ-infected mice at 10 PFU, while a few
v-cyclin.LacZ-infected mice survived longer than any wt
HV68-infected mice at 103 and 106 PFU. Thus,
wt HV68 and v-cyclin.LacZ demonstrated little or no
difference in acute-phase growth in vivo, as determined by measuring
titers of virus in organs of C57Bl/6 mice or virulence in SCID mice,
and as such the v-cyclin gene was deemed dispensable for acute virus
replication in vivo.
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HV68 induces chronic pathology, including aortitis, splenic
fibrosis, and mortality, in IFNR/ mice (21,
71). IFNR/ mice infected with 2 × 107 PFU of wt HV68 or v-cyclin.LacZ were
monitored for survival and histopathologic indications of splenic
pathology and large-elastic-vessel arteritis. Rates of survival of
IFNR/ mice infected with wt HV68 or v-cyclin.LacZ
revealed no significant difference in virus-induced mortality over the
course of three independent experiments (Fig.
4A). Analysis of splenic pathology also
demonstrated no detectable difference in fibrosis/atrophy between mice
infected with wt HV68 and v-cyclin.LacZ-infected mice
(data not shown). Similarly, the incidence of arteritis in the great
elastic vessels in IFNR/ mice infected with wt
HV68 (80%; 31 of 39 infected mice) did not differ significantly
from that of v-cyclin.LacZ-infected mice (84%; 32 of 38 infected mice). However, microscopic analysis of the arteritic lesions
revealed a slight difference in severity of the arteritic lesions (see
Materials and Methods). Aortic lesions in
v-cyclin.LacZ-infected IFNR/ mice were
slightly less severe than those in wt HV68-infected IFNR/ mice (Fig. 4B) (P = 0.0326).
It should be noted that the difference between histology scores of 3 and 5 is the extent of neutrophilic infiltration and necrosis in the
media of the aorta (Dal Canto et al., submitted). Thus, the slight
decrease in severity of the aortic lesions induced by
v-cyclin.LacZ indicates that this mutant virus has a subtle
defect in the induction of chronic pathology in the great elastic
vessels.
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The HV68 v-cyclin is dispensable for establishment of latency in
vivo but is critical for reactivation from latency.
The
experiments detailed above demonstrated that the v-cyclin is not
required for acute or persistent virus replication in vivo. We next
examined the ability of v-cyclin.LacZ to establish latency in
vivo. HV68 establishes latency in macrophages and B cells
(72), and both PECs and splenocytes harbor latent virus (73). To monitor the establishment of latency, we measured
the frequency of viral-genome-positive cells in PECs and
splenocytes 42 days postinfection, using a limiting-dilution
nested-PCR assay capable of detecting a single copy of the viral genome
in a background of 104 uninfected cells (72,
73). As discussed below, acute virus replication was cleared from
the spleen and peritoneum by 16 days postinfection (70).
Splenocytes isolated from wt HV68-, v-cyclin.MR-, or
v-cyclin.LacZ-infected C57Bl/6 mice contained equivalent
frequencies of viral-genome-positive cells (~1 in 4,000 cells) (Fig.
5A). C57Bl/6 PECs from wt HV68-,
v-cyclin.MR-, or v-cyclin.LacZ-infected mice also
demonstrated very similar frequencies of viral-genome-positive cells
(~1 in 3,000 cells for wt HV68 and v-cyclin.MR and ~1 in 4,000 cells for v-cyclin.LacZ) (Fig. 5B). Therefore, the
ability of the v-cyclin.LacZ to establish latency was not
significantly different from that of wt HV68 in either splenocytes
or PECs of infected C57Bl/6 mice.
|
HV68, v-cyclin.MR, and
v-cyclin.LacZ resulted in equivalent establishment of latency, we
determined the abilities of these infected cells to reactivate the
virus from latency in an ex vivo reactivation assay (70, 72,
73). Splenocytes and PECs were analyzed by limiting-dilution
reactivation analysis on MEF indicator cells (Fig. 5C and D). Viral CPE
on the MEF monolayers was scored 21 days after explanation. Notably, splenocytes isolated from wt HV68- or v-cyclin.MR-infected
C57Bl/6 mice exhibited a low but detectable frequency of viral
reactivation (~1 in 200,000 cells) (Fig. 5C). However, no
reactivation was observed with splenocytes harvested from
v-cyclin.LacZ-infected C57Bl/6 mice (Fig. 5C). This impairment
in v-cyclin.LacZ reactivation was readily apparent in latently
infected PECs, for which the frequency of virus reactivation for both
wt HV68 (~1 in 15,000 cells)- and v-cyclin.MR (~1 in
25,000 cells)-infected mice was easily measured, while there was only a
very low level of detectable virus reactivation for PECs harvested from
v-cyclin.LacZ-infected mice (<1 in 106 cells)
(Fig. 5D). Thus, the v-cyclin.LacZ virus was severely impaired
for reactivation from latency in normal C57Bl/6 mice.
We have previously reported that HV68-infected B-cell-deficient mice
have altered latency (70, 73). These mice have a slightly
elevated (approximately sixfold) frequency of viral-genome-positive cells in PECs compared to C57Bl/6 control mice. In the spleen, the frequencies of viral-genome-positive cells in B-cell-deficient and
C57Bl/6 control mice are very similar. However, the frequency of
HV68 reactivation in splenocytes and PECs of infected
B-cell-deficient mice was 50- to 100-fold higher than that for infected
C57Bl/6 mice at day 42 postinfection.
To determine whether the v-cyclin plays an important role in virus
reactivation in these immunocompromised mice, we compared the
frequencies of viral-genome-positive PECs and splenocytes and the
frequencies of cells reactivating virus in B-cell-deficient mice
infected with wt HV68, v-cyclin.MR, or
v-cyclin.LacZ. In B-cell-deficient mice at day 42 postinfection, the frequencies of v-cyclin.LacZ genome-positive
cells in the spleen (~1 in 5,000 cells) and PECs (~1 in 4,500 cells) were slightly lower than the frequencies observed in mice
infected with wt HV68 (spleen, ~1 in 1,500 cells; PECs, ~1 in
700 cells) or v-cyclin.MR (spleen, ~1 in 2,200 cells; PECs,
~1 in 1,200 cells) (Fig. 6A and B).
Consistent with the results obtained in infected C57Bl/6 mice, the
frequency of splenocyte virus reactivation was significantly lower in
B-cell-deficient mice infected with v-cyclin.LacZ virus (~1
in 200,000 cells) than in those infected with either wt HV68 (~1
in 30,000 cells) or v-cyclin.MR (~1 in 50,000 cells) (Fig.
6C). A greater reduction in reactivation efficiency was observed in
PECs harvested from B-cell-deficient mice infected with
v-cyclin.LacZ (~1 in 150,000 cells) than in mice infected
with wt HV68 (~1 in 1,800 cells) or v-cyclin.MR (~1 in
2,500 cells). These results demonstrated that the phenotype of the
v-cyclin mutation was dominant over the enhanced-reactivation phenotype
observed in B-cell-deficient mice. In addition, the decreased
reactivation efficiency of the v-cyclin mutant virus in
B-cell-deficient mice indicates that the reactivation defect is
apparent in a latently infected non-B-cell population, which is almost
exclusively macrophages in PECs of B-cell-deficient mice
(72).
|
HV68 and v-cyclin.LacZ, was used to
infect C57Bl/6 mice, and PECs were analyzed in the ex vivo reactivation
assay 28 days postinfection (Fig.
7). As observed with the
v-cyclin.LacZ virus, there was a ~100-fold reduction in the
frequency of reactivation of v-cyclin.stop or v-cyclin.LacZ in
PECs compared to wt HV68. Thus, this analysis provides strong
evidence that the observed defect in virus reactivation maps to the
v-cyclin gene.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
All known gamma-2 herpesviruses encode a v-cyclin that is well
conserved in sequence and position in the viral genome. Here we have
demonstrated that the v-cyclin is critical for reactivation of HV68
but is dispensable for acute virus replication in vivo. Based on this,
we propose a model in which latently infected cells that have exited
the cell cycle (e.g., resting B cells and macrophages) require v-cyclin
function to reenter the cell cycle, thus poising them at a stage of the
cell cycle which is permissive for virus replication. This model is
consistent with our current understanding of v-cyclin expression in
HV68 infection based on two studies that have analyzed v-cyclin mRNA
expression in latency. v-cyclin transcription was detected by reverse
transcription-PCR in 4 of 16 and 1 of 16 reactions using RNA harvested
from latently infected PECs or splenocytes, respectively
(68). v-cyclin expression was not detected by Northern
analysis of uninduced S11 cells, a B-lymphoma cell line harboring
episomal HV68 (30). The v-cyclin protein is expressed in
ex vivo reactivating cultures (data not shown); however, the small
number of latently infected cells and the asynchronous nature of
reactivation do not allow for clear differentiation of expression in
latently infected cells, in early reactivating cells, or in the
subsequent spread and lytic infection in this system. The v-cyclin is
clearly expressed during lytic infection and in reactivation and may be
expressed at a low level in some latently infected cells. The inability
of cellular cyclin/cdk inhibitors to inhibit the KSHV and HVS v-cyclins
(24, 41, 65) raises the possibility that the v-cyclins have
evolved to efficiently activate cdk's, even in resting differentiated
cells (61). However, the data presented here also
demonstrate that this function of the v-cyclin is clearly dispensable
for direct entry into the lytic cycle during virus replication in
permissive cells in vitro and in vivo. This difference between the
requirements for reactivation and those for direct entry into the lytic
cycle may reflect (i) differences in the cell types infected, (ii) the presence of a virus-encoded redundant activity which functions during
direct entry into the lytic cycle but is not appropriately expressed
during virus reactivation, and/or (iii) acute virus replication in vivo
primarily occurring in cycling cells.
Is the v-cyclin gene critical for reactivation from latency in other gamma-2 herpesviruses? Despite the conservation of the gamma-2 herpesvirus v-cyclins, there are differences in their expression. The HVS v-cyclin protein is expressed in transformed T cells (32), and the KSHV v-cyclin transcript is expressed in KSHV-positive cell lines established from peripheral effusion lymphomas (11, 12, 14, 16). However, the KSHV v-cyclin was not detected in one case of benign, localized Castleman's disease (40), indicating that v-cyclin expression may vary in B cells latently infected with KSHV.
The KSHV v-cyclin is expressed as part of a complex transcriptional unit. The KSHV v-cyclin gene is flanked by gene 73, which encodes a nuclear antigen expressed in the spindle cells of Kaposi's sarcoma [latency-associated nuclear antigen (LANA)] (11, 35, 50), and K13, which encodes an inhibitor of FLICE (v-FLIP) (52). The ORFs encoding LANA 73, v-cyclin, and v-FLIP are expressed under the control of a single promoter, resulting in at least three distinct alternatively spliced transcripts which are 3' coterminal (16). There is a slight increase in the level of KSHV v-cyclin-specific mRNA upon phorbol ester induction of lytic-gene expression in some PEL cell lines (16).
In contrast to v-cyclin expression by KSHV, HV68 lytic infection of
murine fibroblasts results in strong transcription of the v-cyclin gene
(66). Notably, in the HV68 genome the M11 gene (encoding
v-bcl2) is interposed, in the opposite orientation, between gene 73 and
the v-cyclin gene (gene 72) (Fig. 1A) (67), suggesting that
the v-cyclin gene and gene 73 are regulated independently (67). While HV68 v-cyclin is abundantly transcribed
during lytic infection, we previously detected little transcription of gene 73 or M11 during virus replication in NIH 3T12 fibroblasts (68), although others have detected gene 73 and M11
transcripts in poly(A)+ RNA from lytically infected BHK
cells (62). There are other examples of genes with
contrasting expression patterns in HV68 and KSHV. The KSHV genes
encoding GPCR and v-bcl2 are induced by phorbol ester and are therefore
considered to be lytic genes (55) (reviewed in reference
57), whereas in HV68 infection the GPCR (gene 74)
and v-bcl2 (M11) transcripts are difficult to detect in lytically
infected fibroblasts and score as latency-associated genes in a reverse
transcription-PCR screen of latently infected tissue (68).
However, it is important to note that the tissue culture systems used
to characterize KSHV and HV68 gene expression are quite distinct. In
the case of KSHV, studies have been limited to characterizing phorbol
ester induction of lytic genes in a limited number of latently
infected tumor cell lines, while the characterization of
HV68 lytic-gene expression has been largely limited to virus
replication in permissive fibroblast cell lines. There may be
fundamental differences in the regulation of specific viral genes
depending on the cell type infected and on whether reactivation from
latency or direct entry into the lytic cycle is assessed. In addition,
some genes associated with latency may be induced during virus
replication, as has been observed for the EBV v-bcl2 gene homolog (the
BHRF1 gene) (4). Thus, a great deal of caution must be taken
in inferring the role(s) of a viral gene in pathogenesis based on its
pattern of expression in tissue culture models. The results presented
here confirm this point. Despite scoring as a lytic cycle gene in
permissive fibroblasts, the HV68 v-cyclin gene is not essential for
viral replication but is key for reactivation from latency.
The requirement for the HV68 v-cyclin for reactivation from latency
may well be related to a need to cause cell cycle progression to the
G1/S boundary in order to foster efficient DNA replication during virus reactivation. There is substantial precedence in other
herpesvirus systems for control of cell cycle progression. With respect
to host cyclin function, B-cell growth transformation by EBV is
associated with upregulation of host D-type cyclins through expression
of latency-associated membrane protein 1 (3, 28). The
immediate-early protein ICP0 of the alphaherpesvirus herpes simplex
virus type 1 interacts with host cyclin D3 (34), and mutants
lacking functional ICP0 are complemented by G1/S-phase cellular factors (7). In addition, replication of both
herpes simplex virus and HCMV, a betaherpesvirus, is decreased by drugs that inhibit cdk's (6, 59).
There is also growing evidence that during herpesvirus replication, lytically infected cells are arrested at a specific stage of the cell cycle. In EBV-growth-transformed B cells, the immediate-early transcription activator Zta triggers viral reactivation and also blocks cell cycle progression prior to S phase (10). Similarly, the alphaherpesvirus bovine herpesvirus 1 appears to inhibit cell cycle progression through S phase (58). Infection with the betaherpesvirus HCMV arrests cells at G1/S (and perhaps G2/M) (reviewed in references 17 and 53). Thus, control of the cell cycle, by induction of cell cycle progression and/or arrest at S phase, appears to be an important feature of alpha-, beta-, and gamma-herpesvirus replication.
Given that the HV68 v-cyclin is expressed as an early-late gene in
murine fibroblasts, it is surprising that the v-cyclin is completely
dispensable for acute virus replication in vivo. This suggests that
HV68 encodes other functions that regulate the cell cycle in
permissive cell types in vivo. However, the data presented here clearly
document that the v-cyclin is critical for efficient reactivation from
latency. The latter observation provides the first demonstration of a
viral protein that appears to be specifically required for efficient
reactivation from latency, and it suggests that the molecular events
involved in virus reactivation in a latently infected cell are distinct
from those involved in direct entry into the lytic cycle in a
permissive cell type. It remains to be determined whether the v-cyclins
encoded by other gamma-2 herpesviruses play a similar role during
reactivation. This currently cannot be addressed for KSHV, but it could
be determined for infection of primates with either HVS or the recently
identified rhesus monkey rhadinovirus. Finally, the data presented here
underscore the limitations of in vitro analyses of viral mutants and
the importance of pathogenesis studies in determining the roles of specific herpesvirus genes.
| |
ACKNOWLEDGMENTS |
|---|
S.H.S. was supported by NIH grants CA43143, CA52004, CA58524, CA74730, and HL60090. H.W.V. was supported by grant RPG-97-134-01-MBC from the American Cancer Society and by NIH grants AI39616, CA74730, and HL60090. L.V.D. was supported by grant PF-4379 from the American Cancer Society and grant 5 T32 AI07163 from the NIH.
We thank Avril Adelman of the Washington University Medical Center Biostatistics Department for help with statistical analyses. We also thank members of the laboratories of H.W.V., S.H.S., David Leib, and Lynda Morrison for constructive comments on this research.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Pathology and Immunology, Washington University School of Medicine, Box 8118, 660 S. Euclid Ave., St. Louis, MO 63110-1093. Phone: (314) 362-9223 (H.W.V.) or (314) 362-0367 (S.H.S.). Fax: (314) 362-4096. E-mail: virgin{at}immunology.wustl.edu or speck{at}pathbox.wustl.edu.
| |
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Journal of Virology, August 2000, p. 7451-7461, Vol. 74, No. 16
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