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Journal of Virology, January 1999, p. 404-410, Vol. 73, No. 1
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Human Cytomegalovirus Inhibits Transcription of
the CC Chemokine MCP-1 Gene
Alec J.
Hirsch and
Thomas
Shenk*
Howard Hughes Medical Institute, Department
of Molecular Biology, Princeton University, Princeton, New Jersey
08544-1014
Received 1 July 1998/Accepted 13 October 1998
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ABSTRACT |
In primary human diploid fibroblasts, infection with an unpurified
stock of human cytomegalovirus induced accumulation of the CC chemokine
MCP-1 in the cell culture medium. By 24 h postinfection, the level
of MCP-1 returned to that in uninfected cultures. When cells were
infected with UV-inactivated human cytomegalovirus, the induction of
MCP-1 was still observed, but no reduction was seen by 24 h
postinfection or later. This effect was the result of a decrease in the
level of MCP-1 mRNA present within the infected cell. Infection with
purified virus revealed that the induction of MCP-1 was due to an
activity found in the medium of infected cells; purified virions did
not induce the expression of MCP-1. However, infection with purified
virions repressed the level of MCP-1 mRNA below that found in
uninfected cells. Additionally, infection with human cytomegalovirus
prevented the induction of MCP-1 expression by tumor necrosis factor
alpha and interleukin-1
. The CC chemokine receptor encoded by the
human cytomegalovirus US28 open reading frame (ORF) did not appear to
play a role in this process, since a mutant virus in which the US28 ORF
had been deleted downregulated MCP-1 in the same manner.
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INTRODUCTION |
Human cytomegalovirus (HCMV) is a
ubiquitous human pathogen. HCMV infection is associated with several
clinical manifestations, including CMV mononucleosis, birth defects
when infection occurs in pregnant women, and a variety of clinical
syndromes in immunocompromised and immunosuppressed individuals (for a
review, see reference 6). HCMV has been shown to
exert a variety of effects on the gene expression of the infected host
cell, including the induction of cellular transcription factors by
viral attachment to the cell surface (37), modulation of the
cell cycle with characteristic changes in gene expression (5, 9,
18), and direct effects on host cell promoters by viral gene
products (16, 19). Some of the genes that have been shown to
be induced by HCMV are likely to be involved in the host response to
viral infection. These include several members of the cytokine family
and several members of a subclass of cytokines, the chemokines.
The chemokines are a group of cytokines that exhibit chemotactic
activity for a variety of leukocytes (for reviews, see references 2 and 28). They are divided into
four classes, CXC, CC, C, and CX3C, based on the
arrangement of conserved cysteine residues near the N terminus of the
protein. Chemokines are expressed by a variety of cell types, including
monocytes, lymphocytes, epithelial cells, and fibroblasts. Expression
of the chemokines has been shown to be induced by a variety of stimuli,
including other cytokines, bacterial endotoxins, and viral infection.
Because of their chemoattractant activity for leukocytes, it is
believed that the chemokines are mediators of the inflammatory process
and therefore play an important role in the resolution of viral
infection. Consistent with this view, a mouse with a homozygous
deletion of the gene for the CC chemokine MIP-1
has been shown to
exhibit a dramatically reduced inflammatory response to coxsackievirus
and influenza virus and to experience delayed viral clearance
(8). Additionally, these mice show reduced natural killer
(NK) cell-mediated inflammation in the liver during murine CMV
infection (29).
Several lines of evidence suggest that chemokines may play a role in
HCMV infection. HCMV has been shown to induce the expression of the CXC
chemokine interleukin-8 (IL-8) and the CC chemokine RANTES (10,
22, 23). In addition, elevated levels of the CC chemokine MCP-1
have been detected in the cerebrospinal fluid of human immunodeficiency
virus-infected patients with CMV encephalitis (4). MCP-1 has
been demonstrated to be induced by tumor necrosis factor alpha
(TNF-) and IL-1, cytokines that have been shown to be stimulated
by HCMV (10, 27, 32). Furthermore, the HCMV open reading
frame (ORF) US28 encodes a putative seven-transmembrane-domain G-protein-coupled receptor that has been shown to be a functional receptor for MCP-1, RANTES, MIP-1, and MIP-1 (12, 15,
24).
In this report, we examine the effect of HCMV on the expression of the
CC chemokine MCP-1. We have found that an unpurified stock of HCMV
strongly induces MCP-1 mRNA and protein expression. This induction is
not a direct effect of the virus but appears to be due to a factor that
is secreted into the cell culture medium by HCMV-infected cells.
Additionally, HCMV acts to inhibit MCP-1 expression at the level of
transcription during infection. This transcriptional repression occurs
at early times postinfection (p.i.) and requires viral gene expression.
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MATERIALS AND METHODS |
Cells and viruses.
Primary human foreskin fibroblasts (HFFs)
were maintained in Dulbecco's modified Eagle medium (DMEM)
supplemented with 10% fetal bovine serum. HFFs were used between
passages 7 and 14 and infected with HCMV at a multiplicity of infection
(MOI) of 3 PFU/cell 3 to 4 days after reaching confluence. To avoid
cell stimulation by fresh serum, cells were returned after infection to
the culture medium in which they were previously maintained.
Supernatants of infected cells for use as viral stocks were obtained by
infecting HFFs at an MOI of 0.1 with HCMV laboratory strain AD169. Five
to seven days after cells showed 100% cytopathic effect, the medium
was harvested and cellular debris was removed by centrifugation at
8,000 × g for 20 min at 4°C. Stock titers were
determined by plaque assay.
Viral particles were purified by velocity centrifugation in a
D-sorbitol gradient as previously described
(
31). Briefly,
30 ml of an infected-cell supernatant
prepared as described above
was underlayed with a 7-ml sorbitol cushion
containing 20%
D-sorbitol,
50 mM Tris-HCl (pH 7.2), 1 mM
MgCl
2, and 100 µg of bacitracin/ml
in an Ultra-Clear
centrifuge tube (Beckman). Virions were pelleted
by centrifugation at
55,000 ×
g for 1 h at room temperature. Pellets
were resuspended in DMEM to produce a purified virus
stock.
To separate the MCP-1-inducing activity of an unpurified viral stock
from the virus, the stock was filtered through a 100-kDa-cutoff
membrane in a stirred cell concentrator (model 8200; Amicon, Inc.).
Virus stocks were UV inactivated by placing 2 to 5 ml of an unpurified
stock or a purified stock resuspended in 2 to 5 ml
of serum-free DMEM
in a 15-cm-diameter dish and irradiating with
UV light for 15 min at 2 J/m
2 per s as previously described (
38).
Northern blotting.
Total RNA was isolated from HFFs by using
TRIzol reagent (Gibco BRL), subjected to electrophoresis in
formaldehyde-containing agarose gels, and transferred to a nylon
membrane as described previously (1). Four micrograms of
membrane-bound RNA was probed with cDNA probes labeled by random
priming in the presence of 32P. The MCP-1 cDNA probe was
obtained from the I.M.A.G.E. Consortium (Genome Systems). The RANTES
cDNA was obtained by reverse transcription of RNA obtained from
12-O-tetradecanoylphorbol13-acetate (TPA)-differentiated THP-1 cells with RANTES-specific oligonucleotide primers. A cDNA for
one of the cytosolic phospholipases A2, cPL2 (39), which is
not transcriptionally induced during HCMV infection (38), was used as a loading control for all Northern blots. Where indicated, TNF- (R&D Systems) was added to cells at a final concentration of 10 ng/ml. IL-1 (R&D Systems) was added to a final concentration of 1 ng/ml. To inhibit viral DNA replication, phosphonoacetic acid (PAA) was
added to a final concentration of 100 µg/ml 1 h after infection.
To block viral attachment, heparin was added to cells at 10 µg/ml
before the addition of virus (7).
ELISA and immunoprecipitations.
Medium was collected from
infected HFF cultures, and the supernatant was assayed by enzyme-linked
immunosorbent assay (ELISA), using a Quantikine plate specific for
human MCP-1 (R&D Systems) in accordance with the manufacturer's protocol.
For immunoprecipitations, confluent 10-cm-diameter dishes of HFFs were
labeled with [
35S]methionine for 1 h and lysed in
0.5 ml of lysis buffer (50 mM
Tris-Cl [pH 8.0], 150 mM NaCl, 0.1%
sodium dodecyl sulfate, 1.0%
Nonidet P-40, 0.5% sodium deoxycholate,
100 µg/ml phenylmethylsulfonyl-fluoride,
complete protease inhibitor
cocktail tablet [Boehringer Mannheim
Biochemicals] [1 tablet/50 ml
of buffer]). Radioactivity in 5-µl
samples of lysates was quantified
in a Beckman scintillation counter
(model LS5000TD). Equal amounts of
radioactivity were used in
a total volume of 0.25 ml for each
immunoprecipitation. Immunoprecipitations
were carried out with
anti-MCP-1 monoclonal antibody 5D3-F7 (Pharmingen)
(
26), and
complexes were captured with protein A-Sepharose beads.
Precipitates
were washed in lysis buffer, and proteins were separated
on a 10%
polyacrylamide gel containing sodium dodecyl sulfate.
Proteins were
visualized by
autoradiography.
Nuclear run-on assays.
Approximately 4 × 107 HFFs were used for each determination. Where indicated,
cells were infected at an MOI of 3 PFU/cell and treated with IL-1 at
a final concentration of 1 ng/ml. Cells were harvested at 24 h
p.i., which was 1.5 h after IL-1 addition. Nuclei were prepared
and run-on transcription was performed as described elsewhere
(1).
Construction of ADsubUS28 virus.
Cosmid pCM1035
(11) was digested with EcoRI and ClaI,
and the 3.5-kb fragment corresponding to HCMV strain AD169 nucleotides 218438 to 221952 was isolated and cloned into pSP72 (Promega). This
fragment contains the US28 ORF (nucleotides 219200 to 220256) and
approximately 1 kb of flanking sequence on either side. This plasmid
was digested with SacII and StuI, which cut
within the US28 ORF (at positions 219222 and 219629, respectively), and
the US28 sequences were replaced by the enhanced green fluorescent protein (EGFP) ORF (from plasmid EGFP-N1 [Clontech]), regulated by
the simian virus 40 (SV40) early promoter and containing an SV40
poly(A) site, to create psubUS28. The pSP72 background sequences were
removed from psubUS28, and 5 µg of the fragment containing the HCMV
and GFP sequences was transfected with 2 µg of AD169 DNA and 2 µg
of the pp71 expression plasmid pCMV-pp71 (3) into HFFs.
Green fluorescent plaques were isolated, and the substituted virus was
propagated and plaque purified. DNA was isolated from infected cells,
digested with BamHI, and analyzed by Southern blotting as
described elsewhere (1) to confirm the structure of the
mutant virus, ADsubUS28.
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RESULTS |
Induction and subsequent repression of MCP-1 protein levels in
cells infected with crude, unpurified HCMV stocks.
Initially, we
examined the effect of HCMV infection on cellular expression of MCP-1.
Primary HFFs were infected with an unpurified stock of HCMV at an MOI
of 3 PFU/cell. The cells were washed at various times and fed with
fresh, serum-free medium. At the end of a 4-h interval, the medium was
collected and the MCP-1 protein concentration was measured by a
sandwich ELISA (Fig. 1A). During the 0- to 4-h interval, approximately fourfold more MCP-1 accumulated in the
culture medium of infected cells than in that of uninfected cells.
Between 4 and 8 h p.i., infected cells continued to secrete more
MCP-1 than did uninfected controls, but at 20 to 24 h and 44 to
48 h p.i., little MCP-1 was secreted by HCMV-infected cells.
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FIG. 1.
MCP-1 concentration in the culture medium of infected
fibroblasts. (A) MCP-1 secreted into the culture medium during 4-h
intervals. HFFs were infected with active (CMV) or UV-inactivated
(UV-CMV) HCMV at an MOI of 3 PFU/cell. At the indicated times, fresh,
serum-free DMEM was added, and supernatants were collected 4 h
later. MCP-1 concentration in the medium was measured by ELISA. (B)
Total MCP-1 secreted into the culture medium during infection.
Supernatant was collected at the indicated times p.i., and the MCP-1
concentration was measured by ELISA. All values shown are the averages
of data from three independent experiments. mock, supernatant from
mock-infected cells.
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When the infectivity of the viral stock was destroyed by exposure to UV
light prior to infection, induction of MCP-1 similar
to that seen with
active virus was observed between 0 and 4 h
(Fig.
1A). However,
during the 4- to 8-h time interval, significantly
more MCP-1 was
secreted by cells treated with inactivated virus
than by those treated
with active virus. Furthermore, between
20 and 24 h p.i. and
between 44 and 48 h p.i., cells treated with
the UV-inactivated
virus secreted levels of MCP-1 that were approximately
25-fold higher
than those secreted by uninfected or HCMV-infected
cells.
We also measured the total MCP-1 that accumulated in the medium over
the course of infection. HFFs were infected as described
above, and
medium was harvested at various time points. Before
infection, the
MCP-1 concentration in the medium was approximately
5 ng/ml. In cells
infected with active HCMV, the increase in MCP-1
above this background
level was insignificant (Fig.
1B). In contrast,
infection of cells with
UV-inactivated HCMV resulted in a dramatic
increase in the MCP-1
concentration in the culture medium. This
increase was clearly observed
by 24 h p.i. By 48 h p.i., MCP-1
levels were approximately
10-fold higher in cultures infected
with inactivated virus than in
uninfected cultures or in the medium
of HCMV-infected cells (Fig.
1B).
It has been reported previously that RANTES accumulates within cells
late in infection but is not secreted into the medium
(
22).
To determine if MCP-1 protein is sequestered within infected
cells,
HFFs were infected with HCMV or UV-inactivated HCMV and
labeled with
[
35S]methionine, and MCP-1 was immunoprecipitated from
cellular extracts
by using an anti-MCP-1 monoclonal antibody. As shown
in Fig.
2,
at 8 h p.i., the antibody
precipitated an approximately 10-kDa
protein, consistent with the
reported size of the unglycosylated
precursor form of MCP-1. Similar
levels of MCP-1 were present
in cells infected with HCMV and
UV-inactivated HCMV at this time
point. At 24 h p.i., however,
MCP-1 was detected only in cells
treated with UV-inactivated HCMV,
indicating that MCP-1 protein
is not sequestered within the cells
infected with the active virus
but rather is almost completely absent
by 24 h p.i.
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FIG. 2.
MCP-1 within infected cells. HFFs were infected with
HCMV or UV-inactivated HCMV (UV HCMV). 35S-labeled proteins
were immunoprecipitated by an antibody (Ab) specific to MCP-1, and the
immunoprecipitate was analyzed by electrophoresis and autoradiography.
The positions to which marker proteins migrated are indicated on the
left (in kilodaltons), and bands corresponding to the chemokine are
labeled MCP-1. M, mock-infected cells.
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MCP-1 mRNA levels change in concert with protein levels.
We
next examined the effect of HCMV infection on the level of MCP-1 mRNA.
HFFs were infected at an MOI of 3 PFU/cell, and RNA was prepared at
various times after infection and analyzed by Northern blotting. The
level of MCP-1 mRNA initially rose after infection, reaching a peak by
approximately 6 h p.i., and declined during later time periods
(Fig. 3A). When cells were exposed to the
UV-inactivated virus stock, MCP-1 mRNA was induced with similar kinetics but remained elevated through 72 h p.i. (Fig. 3B).
Because MCP-1 protein levels change in concert with the levels of mRNA, we believe that the regulation of MCP-1 by HCMV occurs primarily at the
RNA level.
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FIG. 3.
MCP-1 mRNA accumulation during infection. HFFs were
infected by HCMV (A) or UV-inactivated HCMV (B). RNA was collected at
the indicated times and analyzed by Northern blotting with an
MCP-1-specific cDNA probe. A cDNA probe for one of the cellular cPLA2
mRNAs was used as a loading control. Mock, mock-infected cells.
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Induction of MCP-1 mRNA is due to a factor present in the medium of
infected cells.
The experiments described above used an unpurified
virus stock, i.e., an infected-cell supernatant, as the source of
virus. Such a virus stock almost certainly contains various cytokines and other signaling molecules that have been induced during the course
of viral infection. These factors in the virus stock may be responsible
for, or contribute to, the observed induction of MCP-1 mRNA. To
differentiate between direct induction of MCP-1 by the virus and
induction by a contaminating factor, the crude virus stock was purified
by high-speed centrifugation of virions through a sorbitol cushion,
separating the particles from low-molecular-weight contaminants. After
determination of its titer, the purified virus stock was used to infect
HFFs at an MOI of 3 PFU/cell; this MOI was the same as that used for
infection with the unpurified virus stock. As seen in Fig.
4 (compare lanes 1 and 3), the purified virus did not induce MCP-1 mRNA levels at 8 h p.i., even though it
expressed the virus-coded IE1 mRNA. To further characterize the
inducing activity, the unpurified virus stock was filtered through a
100-kDa-cutoff membrane and the flowthrough was added to HFFs (Fig. 4A,
lane 4). The filter effectively removed virus from the medium, as
measured by Northern blotting of the virally encoded IE1 mRNA and by
plaque assay, and the filtered medium retained the ability to induce
MCP-1. In addition, blocking viral attachment to the cells by the
addition of heparin to the crude viral stock did not interfere with
induction of MCP-1 mRNA (Fig. 4, lane 5). Failure to induce expression
of IE1 mRNA confirmed that heparin successfully blocked the infection.
The above-described experiments demonstrated that the observed
induction of MCP-1 RNA during HCMV infection is due to a factor that
accumulates in the medium of infected cells and is not mediated
directly by the viral particle. The inducing factor is present only in
the medium of infected cells, not in that of uninfected cells.
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FIG. 4.
MCP-1 is induced by a factor present in the medium of
infected cells. (A) HFFs were infected and/or treated as described in
the text, and RNA was collected 8 h later and analyzed by Northern
blotting. Lanes: 1, mock-infected cells; 2, crude HCMV stock, MOI = 3; 3, purified HCMV stock, MOI = 3; 4, flowthrough fraction of
crude virus stock filtered through a 100-kDa-cutoff filter; 5, crude
HCMV stock plus heparin (10 µg/ml); 6, mock-infected cells treated
with heparin (10 µg/ml). (B) Purified HCMV stock represses MCP-1 mRNA
levels. Lanes: 1, mock-infected cells; 2, HCMV, 6 h p.i.; 3, HCMV,
24 h p.i.; 4, HCMV, 48 h p.i. The autoradiograph was exposed
for a longer time than in other panels so that the MCP-1 mRNA in the
mock lane would be clearly visible.
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In different experiments we found that the basal level of MCP-1 mRNA
relative to the amount of control mRNAs varied to some
extent. We have
not been able to identify the cause of this variation.
The effect of
viral infection on the level of MCP-1 mRNA was entirely
reproducible in
repeated
experiments.
After infection with purified virions, MCP-1 mRNA levels were reduced
compared to mock-infection levels. The level of MCP-1
mRNA was reduced
below the levels found in uninfected controls
at 24 h p.i. and
later times (Fig.
4B; compare lane 1 with lanes
3 and
4).
HCMV infection interferes with the ability of TNF- and IL-1
to induce MCP-1 mRNA.
We next determined whether purified HCMV
could block the induction of MCP-1 mRNA by two known inducers, TNF-
and IL-1. We observed a marked increase in MCP-1 mRNA at 2 h
after treatment of HFFs with either TNF- (Fig.
5A; compare lanes 1 and 2) or IL-1
(Fig. 5B; compare lanes 1 and 2). To observe the effect of HCMV on this
induction, cells were treated with TNF- or IL-1 for a 2-h
interval at various time points after infection with purified virus.
Infection with HCMV at the time of TNF- treatment (Fig. 5A, lane 3)
did block the induction of MCP-1-specific RNA by TNF-. TNF- could
detectably induce MCP-1-specific RNA through 4 h p.i., but by
8 h p.i., induction was significantly reduced. By 12 h p.i.
and later (Fig. 5A, lanes 7 to 9), TNF- could no longer detectably
induce MCP-1 RNA expression. Similar results were obtained for IL-1
(Fig. 5B), showing that HCMV does not solely block TNF--mediated
effects on MCP-1. The inhibition of IL-1-mediated activation of
MCP-1 does not appear to require the secreted IL-1 receptor
antagonist (IL-1ra), a cellular factor whose expression has been shown
to be increased by HCMV infection (13). When the medium of
infected cells was replaced with fresh medium before the addition of
IL-1, inhibition of MCP-1 was still observed (data not shown),
suggesting that inhibitory factors in the culture medium are not
responsible for the inability of IL-1 to upregulate MCP-1 in
infected cells.
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FIG. 5.
HCMV infection prevents the induction of MCP-1 mRNA
accumulation by TNF- and IL-1. RNA was collected at the indicated
times p.i. and analyzed by Northern blotting with an MCP-1-specific
cDNA probe. A cDNA probe for one of the cellular cPLA2 mRNAs was used
as a control. (A) TNF- treatment. Where indicated, HFFs were treated
with TNF- (10 ng/ml) and infected with HCMV. RNA was collected
2 h after addition of TNF-. Lanes: 1, mock-infected cells; 2, TNF- only; lanes 3 to 9, TNF- added at the indicated times p.i.
(B) IL-1 treatment. IL-1 was added (1 ng/ml) at the indicated
times p.i. (lanes 3 to 9) or alone (lane 2). (C) PAA treatment. HFFs
were infected with HCMV, treated (+) or not treated ( ) with IL-1
or PAA (100 µg/ml, 1 h p.i.) as indicated. RNA was collected at
24 h p.i., 2 h after IL-1 addition.
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The inhibition of IL-1
induction of MCP-1 mRNA occurs in the
presence of the HCMV DNA replication inhibitor PAA (Fig.
5C).
Therefore, this phenomenon does not require viral late gene
expression.
HCMV prevents enhancement of the rate of transcription of MCP-1
mRNA by IL-1.
To determine if the block to MCP-1 mRNA
accumulation occurs at the level of transcription, we performed a
nuclear run-on assay. The addition of IL-1 to HFFs resulted in a
fourfold increase in the rate of transcription (Fig.
6). When cells that had been infected
with purified HCMV were treated with IL-1 at 24 h p.i., when
inhibition of MCP-1 RNA induction was clearly observed, there was no
increase in the rate of transcription (Fig. 6). This result demonstrates that HCMV blocks the induction of MCP-1 mRNA accumulation at the level of transcription. Similar results were obtained with TNF- (data not shown).
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FIG. 6.
HCMV blocks the transcriptional induction of MCP-1 by
IL-1. (A) Nuclear run-on assay of mock-infected HFFs, mock-infected
HFFs treated with IL-1 (1 ng/ml, 1.5 h), and HFFs infected with HCMV
(24 h) and treated with IL-1 (1 ng/ml, 1.5 h). Newly transcribed,
labeled RNAs for MCP-1, -actin, and the viral gene encoding US28
were hybridized to specific cDNAs immobilized on a nitrocellulose
filter. pSP72 is a plasmid lacking a virus-specific insert that was
included to monitor nonspecific binding. (B) Relative rate of MCP-1
transcription after various HFF treatments. The bands in panel A were
quantitated with a phosphorimager, and values are normalized to
-actin. Mock, mock-infected cells; IL-1, treated with IL-1
only; HCMV + IL-1, HCMV infected and treated with IL-1.
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The US28 ORF is not involved in the regulation of MCP-1
expression.
It has been shown that the product of the HCMV US28
ORF is a functional receptor for several CC chemokines, including MCP-1 (12, 15, 24). Because this receptor has been shown to be capable of transducing an intracellular signal in response to ligand
binding, it is conceivable that this receptor is involved in preventing
the induction of MCP-1, perhaps through a negative feedback loop. To
determine whether the US28-encoded chemokine receptor is
involved in the observed regulation of MCP-1 mRNA expression, we
constructed a mutant virus that could not express the US28
product (AdsubUS28). As shown in Fig.
7A, a plasmid construct containing viral
sequences that flank the US28 ORF positioned on either side of a GFP
marker (psubUS28) was used to delete the US28 ORF by homologous
recombination. After several rounds of plaque purification, the
substitution mutant was found to be free of wild-type sequences, as
determined by Southern blot assay (Fig. 7B). A probe corresponding to
the genomic region to the left of the US28 ORF detected a 6.0-kb
fragment in a Southern blot of BamHI-digested AD169 DNA.
After correct recombination of psubUS28 into the viral genome, this
probe detected a 3.9-kb fragment in BamHI-digested viral
DNA. The US28 gene product is not essential for virus growth in tissue
culture; the titers of the mutant and wild-type virus stocks produced
by infection of HFFs at the same input multiplicity were identical
(data not shown).
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FIG. 7.
The US28 gene product is not required for the inhibition
of MCP-1 mRNA induction. (A) Schematic representation of the
US27-US29 region of the HCMV genome, the plasmid construct used to
delete the US28 ORF, and probes used to characterize the mutant
virus. (B) Southern blot of AD169 and ADsubUS28 viral DNA. Viral DNA
was digested with BamHI, resulting in a 6.0-kb fragment
containing the US28 ORF for the wild-type virus and in 3.9- and 2.8-kb
fragments for the mutant, due to a BamHI site within the GFP
marker. (C) Northern blot of RNA isolated from cells infected
with AD169 or the AdsubUS28 mutant and treated with IL-1 (1 ng/ml). RNA was collected at various times p.i. and 2 h after
IL-1 addition. An MCP-1-specific cDNA probe and a cDNA probe for a
cellular cPLA2 RNA (control) were used. Lanes: 1, mock infection; 2, IL-1 only; 3, IL-1 and HCMV (AD169), 4 h p.i.; 4, IL-1
and HCMV (AD169), 24 h p.i.; 5, IL-1 and HCMV (AD169), 48 h p.i.; 6, IL-1 and HCMV (ADsubUS28), 4 h p.i.; 7, IL-1 and
HCMV (ADsubUS28), 24 h p.i.; 8, IL-1 and HCMV (ADsubUS28),
48 h p.i.
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HFFs were infected with AdsubUS28 and treated with IL-1
at various
time points (Fig.
7C). AdsubUS28 prevented the induction
of MCP-1 mRNA
in a manner identical to that of the wild-type virus,
demonstrating
that the US28 ORF is not required for MCP-1 regulation
during
infection.
Regulation of RANTES mRNA expression occurs in a manner distinct
from MCP-1 mRNA regulation.
As mentioned above, the CC chemokine
RANTES has previously been shown to be induced by HCMV infection in a
manner independent of exogenous virally induced cytokines
(22). We detected the induction of RANTES mRNA by Northern
blotting after infection of HFFs with purified HCMV (Fig.
8, lanes 1 to 4), in agreement with the
previous result. We also found that TNF- modestly induced RANTES RNA
expression (Fig. 8, lanes 8 to 10). Interestingly, TNF- and HCMV
appeared to synergize to transiently induce RANTES expression to a much
greater extent (Fig. 8, lanes 5 to 7), and these elevated levels of
mRNA were clearly detected at 8 and 24 h p.i. For comparison,
MCP-1 mRNA levels in the same preparations as those analyzed in the
RANTES blot are shown (middle panel), demonstrating the antagonistic
effect of HCMV on TNF--mediated MCP-1 induction. Again, similar
results were obtained when IL-1 was used in place of TNF- (data
not shown). Thus, HCMV induces RANTES mRNA accumulation while
inhibiting MCP-1 transcription.
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FIG. 8.
HCMV and TNF- synergize to induce RANTES mRNA
accumulation. HFFs were infected with HCMV (MOI = 3) and/or
treated with TNF- (10 ng/ml). RNA was collected at the indicated
times p.i. Northern blots were probed with cDNA specific for RANTES,
MCP-1, or cPLA2. M, mock-infected cells.
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DISCUSSION |
Infection of primary human diploid fibroblasts with HCMV results
in the induction of a secreted activity that can induce MCP-1 mRNA
expression (Fig. 3). We have not yet identified the activity responsible for this induction, but it might include TNF- and/or IL-1, both of which are induced during HCMV infection and are known
to induce MCP-1 (10, 27, 32). During the course of infection, however, the virus prevents induction of MCP-1 mRNA by this
activity (Fig. 3). The prevention of MCP-1 mRNA induction requires
viral gene expression (Fig. 3) and occurs within 8 h after
infection. Elevated levels of MCP-1 protein were secreted into the cell
culture medium during all time intervals when elevated mRNA levels were
present (Fig. 1), and MCP-1 protein is not sequestered within the
infected cell (Fig. 2), as is seen with RANTES, another CC chemokine
(22). The virus can be purified away from the MCP-1-inducing activity (Fig. 4), and the purified virus retains the ability to
prevent induction of MCP-1 by TNF- and IL-1 (Fig. 5). This effect
occurs at the level of transcription (Fig. 6). The US28 ORF, which has
been shown to encode a functional receptor for MCP-1 as well as other
CC chemokines, does not contribute to the observed effects on MCP-1
expression (Fig. 7).
How does HCMV block expression of the MCP-1 gene? The prevention of
MCP-1 accumulation occurs at the level of transcription (Fig. 6) and
clearly requires viral gene expression (Fig. 1 and 3). Additionally,
TNF- and IL-1 are prevented from inducing MCP-1 transcription by
8 h p.i. (Fig. 3), and inhibition of DNA replication does not
prevent this inhibition (Fig. 5C). Therefore, we believe that it is
likely that one or more HCMV gene products produced relatively soon
after infection block the induction of MCP-1 transcription. As yet, the
identity of this putative virus-coded inhibitor(s) is unknown. It might
specifically target MCP-1 transcription or interfere at an earlier
point in the signal transduction pathways of TNF- and IL-1.
The MCP-1 promoter has been shown to contain binding sites for the
transcription factors NF-
B and Sp1 (33, 34). NF-B activation has been shown to be crucial for activation of this promoter
by TNF-. Interestingly, it has been shown that HCMV infection
induces the activity of both of these transcription factors (14,
37). However, infection with a purified stock of HCMV does not
stimulate transcription of MCP-1 (Fig. 4A and B), even though NF-B
and Sp1 are upregulated within 30 min following infection. Of note is
the fact that proinflammatory factors such as RANTES and
cyclooxygenase-2 (Cox-2) have also been shown to be induced by NF-B
(21, 25, 30), and RANTES and Cox-2 are induced during HCMV
infection (22, 38a). It is possible that the inhibitor
postulated above is contained within the viral tegument and introduced
immediately upon infection, blocking the induction of MCP-1, while
other NF-B-responsive genes are induced. If this is the case, the
initial level of this inhibitor within the infected cell must be
insufficient to prevent MCP-1 induction by TNF- or IL-1 (and the
unidentified activity within the crude viral stock) because prevention
of the induction of MCP-1 by these cytokines is not observed until
8 h p.i.
High MCP-1 levels have been reported in the cerebrospinal fluid of AIDS
patients with HCMV encephalitis (4). If our results with
fibroblasts hold true for other cell types, then we would not expect
MCP-1 to be produced by HCMV-infected cells; rather, it would be
produced by cells in the vicinity that would likely respond to TNF-
and IL-1 produced by neighboring infected cells.
It has been shown that MCP-1 is capable of attracting monocytes and NK
cells in vitro and in vivo (17, 20, 35, 36). These cells
play a major role in the inflammatory response to viral infection. It
is not clear why it might be beneficial to the virus to block MCP-1
expression and secretion in infected cells but not in neighboring cells
that may respond to infection-induced TNF- and IL-1 by producing
MCP-1. Perhaps monocytes and NK cells discriminate MCP-1-producing
cells from cells that do not secrete this chemokine. Further, HCMV can
infect monocytes and undergo latency there. Conceivably, the production
of MCP-1 by neighboring cells might serve to lure monocytes to the site
of infection, where they can be infected by HCMV, facilitating the
maintenance and spread of the virus.
| |
ACKNOWLEDGMENTS |
We thank W. Bresnahan, F. Ferrari, C. Patterson, and B. Wing for
commenting on the manuscript and H. Zhu for helpful discussions.
A.J.H. was supported in part by an American Heart Association
predoctoral fellowship (95-FS-05). T.S. is an American Cancer Society
Professor and an Investigator of the Howard Hughes Medical Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Howard Hughes
Medical Institute, Department of Molecular Biology, Princeton
University, Princeton, NJ 08544-1014. Phone: (609) 258-5992. Fax: (609)
258-1708. E-mail: tshenk{at}princeton.edu.
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Abstract
Introduction
