By
From the Center for Immunology and Departments of Pathology and Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110
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Abstract |
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CD4 T cells and interferon (IFN-
) are required for clearance of murine cytomegalovirus
(MCMV) infection from the salivary gland in a process taking weeks to months. To explain the
inefficiency of salivary gland clearance we hypothesized that MCMV interferes with IFN-
induced antigen presentation to CD4 T cells. MCMV infection inhibited IFN-
-induced presentation of major histocompatibility complex (MHC) class II associated peptide antigen by
differentiated bone marrow macrophages (BMM
s) to a T cell hybridoma via impairment of
MHC class II cell surface expression. This effect was independent of IFN-
/
induction by
MCMV infection, and required direct infection of the BMM
s with live virus. Inhibition of
MHC class II cell surface expression was associated with a six- to eightfold reduction in IFN-
induced IAb mRNA levels, and comparable decreases in IFN-
induced expression of invariant
chain (Ii), H-2Ma, and H-2Mb mRNAs. Steady state levels of several constitutive host mRNAs,
including
-actin, cyclophilin, and CD45 were not significantly decreased by MCMV infection, ruling out a general effect of MCMV infection on mRNA levels. MCMV effects were
specific to certain MHC genes since IFN-
-induced transporter associated with antigen presentation (TAP)2 mRNA levels were minimally altered in infected cells. Analysis of early upstream events in the IFN-
signaling pathway revealed that MCMV did not affect activation
and nuclear translocation of STAT1
, and had minor effects on the early induction of IRF-1 mRNA and protein. We conclude that MCMV infection interferes with IFN-
-mediated induction of specific MHC genes and the Ii at a stage subsequent to STAT1
activation and nuclear translocation. This impairs antigen presentation to CD4 T cells, and may contribute to
the capacity of MCMV to spread and persist within the infected host.
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Introduction |
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Human cytomegalovirus (HCMV)1 is a major cause of morbidity and mortality in immunocompromised individuals. Studies using murine cytomegalovirus (MCMV), which serves as a useful animal model for HCMV, have demonstrated that MCMV infection provokes strong responses by both innate and specific arms of the immune system. However, even in the immunocompetent host, MCMV; (a) causes disseminated acute infection; (b) persistently produces infectious virus within the salivary gland for weeks to months after induction of specific immunity; and (c) establishes a life-long latent state. This suggests that the virus is able to evade or modify responses by the immune system.
Multiple components of the innate and specific immune
responses are active during acute MCMV infection. IFN-/
, TNF-
, IL-12, and IFN-
contribute to the control
of MCMV during initial stages of infection (1). NK cells
contribute to control of MCMV infection (6) through production of IFN-
(3, 4) and cytotoxicity (7). Specific immune function is required for protection from virus-induced
mortality (8). CD8 T cells mediate clearance of infectious
virus from most peripheral organs and confer protective immunity (9). However, CD4 T cells can effectively clear
MCMV infection from peripheral organs in the absence of
CD8 T cells (10). Clearance of MCMV from the salivary
gland involves CD4 T cells (11) and IFN-
(1).
Both MCMV and HCMV have evolved mechanisms for
evading CD8 T cells and NK cells. Both viruses inhibit
MHC class I expression on infected cells (12). Similarly,
both MCMV and HCMV interfere with NK cell activity
through the actions of virally encoded MHC class I homologs (17, 18). Since CD4 T cells are also essential for
control of MCMV infection from salivary gland, it is not
surprising that MCMV inhibits priming of CD4 T cells in
vivo (19). However, mechanisms underlying CMV-mediated inhibition of CD4 T cell activation have not been
completely defined. HCMV-induced IFN-, as well as direct infection, inhibits IFN-
-induced MHC class II expression in endothelial cells (20, 21). HCMV also alters
MHC class II expression in cultured human peripheral blood
macrophages (M
s; reference 22). Similarly, MCMV-induced
IFN-
/
inhibits MHC class II expression on M
s during
the innate immune response (23).
Ms are important in the pathogenesis of both HCMV
and MCMV. Key functions of M
s include presentation of
antigen to CD4 T cells via MHC class II and secretion of
cytokines. M
s are a site of MCMV replication in multiple
sites (24). Dissemination of MCMV (and likely HCMV)
to secondary sites of infection is mediated by M
s or M
-like cells within the blood (25). Monocytes and M
s are
a site of long-term latency for HCMV and MCMV (28).
In these studies, MCMV impaired IFN--induced
MHC class II-dependent antigen presentation by bone
marrow (BM)M
s. This effect was due to the failure of
IFN-
to efficiently induce mRNAs for IAb, invariant chain
(Ii), H-2Ma, and H-2Mb in infected cells, whereas quantities of transporter associated with antigen presentation (TAP)2 mRNA were unaltered by infection. This effect on
MHC class II-mediated antigen presentation may contribute to the persistence of MCMV in the host, particularly in
the salivary gland, where CD4 T cells play a critical role.
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Materials and Methods |
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Animals, Media, and BMM Culture.
Viruses and Viral Assays.
MCMV (American Type Culture Collection [ATCC] No. VR-194, Lot 10) was grown, inactivated by UV irradiation, and titered by plaque assay in BALB/ 3T12-3 fibroblasts (ATCC, CCL 164; reference 3). Recombinant MCMV expressing bacterialAnalysis of Cell Surface Protein Expression.
Flow cytometry was performed as previously described (3, 23) on an Epics flow cytometer (Coulter, Miami, FL) using XL analysis software (Coulter) or WinMDI 2.0 (Joseph Trotter, San Diego, CA). IAb high and low BMMSupernatant Transfer Studies.
Wild-type 129 BMMElectrophoretic Mobility Shift Assays.
BMMNorthern Blot Analysis.
Total cellular RNA was harvested from BMMAntigen Presentation Assays.
BMM ![]() |
Results |
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We examined IFN--stimulated presentation of
peptide antigen to CD4 T cells by primary BMM
s after
infection with MCMV. Primary cells were used because
previous studies of MCMV regulation of MHC class I expression had revealed differences between cell lines and primary cells (15). M
s were either mock infected or infected with MCMV, treated with IFN-
for 24 h, fixed, and
tested for the ability to present peptide antigen (Fig. 1 A).
IFN-
treatment of M
s resulted in effective presentation
of
-galactosidase peptide (429-441) to the T cell hybridoma B11, whereas MCMV infection abolished IFN-
-
induced antigen presentation. The antigen presenting cell,
rather than the T cell hybridoma, was impaired by virus since (a) M
s were fixed before incubation with T cells,
and (b) addition of fixed MCMV infected M
s did not efficiently inhibit the capacity of uninfected M
s to present
peptide antigen (Fig. 1 A). Cell surface IAb levels were
evaluated by flow cytometry, since decreased IAb expression might explain defective antigen presentation. MCMV-infected M
s failed to respond to IFN-
stimulation by
upregulation of cell surface IAb (Fig. 1 B).
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MCMV and HCMV infection at a low MOI
impair IFN--induced MHC class II expression via induction of IFN-
/
(20, 23). To evaluate MCMV's effects on
M
responsiveness to IFN-
independently of IFN-
/
,
all experiments were performed at a high MOI in M
s derived from mice carrying a null mutation in the IFN-
/
receptor (IFN-
/
R
/
; reference 32). These M
s do not
respond to IFN-
/
stimulation, but do respond to stimulation with IFN-
as measured by induction of IAb expression (Fig. 1 B and Fig. 2; references 23, 32). Infection of
IFN-
/
R
/
M
s with increasing doses of MCMV resulted in a progressive decrease in the percentage of cells
expressing IFN-
-induced IAb (Fig. 2). Cell viability in the
MCMV-infected M
cultures was >95% at 72 h after infection. Live virus was required for inhibition of IAb expression, since UV-inactivated MCMV, even at a MOI of
6.0, had no effect on M
IAb expression (Fig. 2).
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MCMV-mediated inhibition of IFN--
induced IAb expression was dependent on viral dose (Fig. 2),
which suggested a role for direct infection. To address this
possibility, IFN-
/
R
/
M
s were infected with RM427
(25), a recombinant MCMV expressing
-galactosidase under the MCMV ie1/ie2 promoter/enhancer at an MOI of
0.6, and treated with IFN-
. Cells were fixed 48 h after infection, stained for IAb expression, and separated into IAb
high and low populations by fluorescent activated cell sorting (Fig. 3 A). IAb low M
s were enriched for
-galatosidase positive (infected) cells (Fig. 3, A and B), whereas the
IAb high cells were only 0.5-2.5% positive for
-galactosidase.
This demonstrated that IAb expression is low in MCMV-infected BMM
s. However, over several experiments,
-galactosidase was detected in <100% of IAb low cells,
raising the possibility that a soluble mediator might contribute to low class II expression in
-galactosidase-negative cells.
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To determine whether soluble factors played a role in
MCMV's inhibition of IAb induction in IFN-/
R
/
M
s, supernatant transfer studies were performed. Supernatants from wild-type 129 M
s infected with MCMV or
mock infected for 48 h were ultracentrifuged to remove
free virus and placed on naive M
cultures derived from
either wild-type 129 or IFN-
/
R
/
mice. IFN-
was
added to these secondary cultures, and after 48 h, IAb expression was evaluated (Fig. 4). As expected from our previous work (23), supernatant from MCMV-infected M
s
inhibited IFN-
-induced IAb expression on wild-type
M
s, but not on IFN-
/
R
/
M
s (Fig. 4), confirming
that MCMV-induced IFN-
/
can inhibit IFN-
-induced
MHC class II expression. However, there was no evidence for a soluble mediator affecting IAb expression in MCMV-infected IFN-
/
R
/
M
cultures (Fig. 4). Therefore,
direct infection of the M
, rather than a soluble mediator,
is likely to explain MCMV's effects on MHC IAb expression.
|
IFN- induction of MHC
class II expression occurs at the level of gene transcription
(45), and studies in HCMV-infected endothelial cells have
shown that HCMV infection regulates MHC class II
mRNA levels (21). We therefore evaluated MCMV's effect on mRNA levels of an IFN-
-induced MHC class II
gene, IAb. Infection with MCMV, but not UV-inactivated
MCMV, decreased IFN-
-induced accumulation of IAb
mRNA 6-8-fold at 24 h and 5->10-fold at 48 h after infection (Fig. 5).
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Although a 6-8-fold reduction of IFN--induced IAb
mRNA accumulation within MCMV-infected cells explained a portion of the effects of MCMV infection on antigen presentation (Fig. 1 A), it did not plausibly account
for the at least 30-fold loss of cell surface expression of IAb
(Figs. 1 B, 2, 3, 4). Therefore, we examined the effect of
MCMV on expression of another IFN-
-inducible gene, Ii,
which is essential for efficient cell surface expression of MHC
class II (46, 47). Northern blot hybridization showed that
IFN-
-induced Ii transcript levels are reduced 5->10-fold
at 24 h and >10-fold at 48 h in MCMV-infected BMM
cultures. The combined effect of MCMV infection on
IFN-
-induced IAb and Ii mRNA accumulation plausibly
explains most or all of the decreased MHC class II expression on MCMV-infected M
s.
To address the possibility that MCMV's reduction of IAb
and Ii mRNA levels was due to a general effect on host
mRNA, levels of several constitutive host mRNAs were
evaluated. MCMV infection for up to 48 h did not significantly decrease levels of -actin (Fig. 5, A and B), cyclophilin (see Fig. 8), or CD45 (data not shown) mRNAs as
measured against a 28S RNA loading control. In contrast,
treatment of BMM
s with actinomycin D for 5-10 h revealed diminished
-actin, cyclophilin, and CD45 mRNA
levels compared to untreated cells (data not shown), demonstrating that the half lives of these mRNAs were short
enough for us to detect significant MCMV induced alteration in their stability. Thus, inhibition of IFN-
-induced
IAb and Ii mRNA accumulation is not solely due to a general effect on mRNA levels by MCMV.
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IFN- initiates a cascade of events
that leads to MHC class II mRNA accumulation, any one
of which could, a priori, be altered by MCMV infection.
We examined the effect of MCMV on an essential early
event in IFN-
receptor signaling, activation and nuclear translocation of the latent cytoplasmic transcription factor
STAT1
. Activation of STAT1
is essential for IFN-
induction of MHC class II expression (48). M
s were either
mock infected or infected with MCMV for 1, 24, or 48 h
before stimulation with IFN-
, preparation of nuclear extracts, and evaluation of STAT1
binding activity by
EMSA (Fig. 6). The presence of STAT1
in the activated
complex was confirmed by super-shift with antibody
against STAT1
(Fig. 6), whereas antibody to IRF-1 did
not result in retardation of the complex (data not shown).
IFN-
treatment resulted in comparable levels of STAT1
DNA binding activity in both mock- and MCMV-infected M
s, at 1, 24 (Fig. 6), and 48 (data not shown) h after infection. STAT1
activation was also equivalent in mock-
and MCMV-infected M
s after treatment with IFN-
at
doses as low as 0.5 IU/ml (data not shown). We conclude
that the IFN-
signaling pathway remains intact through
STAT1
activation in MCMV-infected BMM
s.
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To directly
assess the effects of MCMV on the transactivating function
of STAT1, we examined IFN- induction of IRF-1
mRNA and protein. IRF-1 is rapidly induced at the transcriptional level after IFN-
stimulation in a STAT1
-dependent manner (48), with maximal mRNA expression
by 2 h of cytokine treatment (49). M
s were mock infected or infected with MCMV at an MOI of 5.0 for 3 h
and treated with IFN-
(100 IU/ml) for the final 2 h of infection. Northern blot analysis showed that induction of
IRF-1 mRNA accumulation by IFN-
in MCMV-infected
M
s was 70-90% that of uninfected cells (Fig. 7 A). To assess MCMV effects on expression of IRF-1 protein, we assayed nuclear extracts of M
s that were mock or MCMV
infected for a total of 5 h, with IFN-
treatment during the
final 4 h for binding to the IRF-E element of the murine
(2'-5') oligoadenylate synthase promoter (35) by EMSA.
IFN-
treatment induced IRF-1 DNA binding activity in
MCMV-infected cells (Fig. 7 B) as confirmed by super-shift
of the complex after incubation with anti-IRF-1, but not
anti-STAT1
, antiserum. IFN-
-induced IRF-1 DNA
binding activity was decreased by at most 50% in MCMV-infected M
s compared to mock-infected M
s. IRF-1
DNA binding activity could also be induced by IFN-
in
M
s that had been infected with MCMV for 24 h (data not
shown).
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To assess the specificity of the block of IFN- signaling
caused by MCMV infection, we examined levels of several
IFN-
-induced transcripts in addition to IAb, Ii, and IRF-1. IFN-
/
R
/
M
s were infected with MCMV or
UV-inactivated virus, or mock infected for 24 or 48 h in
the presence or absence of IFN-
, and mRNA levels were
analyzed by Northern blot hybridization. IFN-
induction
of H-2Ma and H-2Mb expression was significantly inhibited by MCMV at 24 h (Fig. 8), whereas IFN-
-induced
TAP1 mRNA levels were decreased to a lesser extent.
However, TAP2 mRNA levels were not significantly decreased by MCMV infection (Fig. 8). Northern blot data
from 48-h infections were similar to that of 24-h infections (data not shown). The lack of effect on IFN-
induction of
TAP2 transcript levels by MCMV demonstrated that
MCMV selectively affects expression of a subset of IFN-
-
inducible genes.
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Discussion |
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CD4 T cells are required for clearance of salivary gland MCMV infection (11), yet infectious virus is found in the salivary gland for many weeks after acute infection, suggesting that MCMV antagonizes CD4 T cell function in some manner. It has also been shown that MCMV impairs CD4 T cell priming in vivo (19). These observations led us to examine the effects of MCMV on presentation of antigen to CD4 T cells and induction of genes associated with MHC class II cell surface expression.
MCMV inhibited IFN- induction of IAb and Ii gene
expression in IFN-
/
R
/
BMM
s, resulting in defective antigen presentation to CD4 T cells. Inhibition of IAb
expression required direct infection of the M
s with live
virus. Early IFN-
signaling events including STAT1
activation and IRF-1 induction remained largely intact in
MCMV-infected cells. Further analysis showed that
MCMV infection altered the induction of multiple genes
encoding proteins important for MHC class II expression and/or presentation of peptide antigen to CD4 T cells,
while another IFN-
-inducible MHC gene was largely
unaffected.
MCMV impaired the ability of IFN- to induce expression
of mRNAs encoding multiple genes important for antigen
presentation to CD4 T cells. These genes included the
MHC class II allele IAb, as well as the Ii p33 subunit and
the peptide-loading protein subunits H-2Ma and H-2Mb.
Although the 6-8-fold reduction of IFN-
-induced IAb
mRNA levels observed in the setting of MCMV infection
undoubtedly contributed to loss of cell surface expression
of IAb (e.g., Fig. 1 B), it was possible that additional factors
accounted for the near complete inhibition of IAb expression. Ii is required for maximal cell surface expression of
MHC class II proteins (46, 47), and induction of Ii by IFN-
was significantly inhibited in MCMV-infected cells. Decreased expression H-2Ma and H-2Mb might also contribute to impaired MHC class II peptide presentation, since
these proteins are necessary for optimal loading of peptide
onto MHC class II molecules (50). However, loss of
H-2Ma and H-2Mb expression probably did not contribute to decreased detection of cell surface IAb, as the antibody 25-9-17S detects IAb loaded with the class II Ii-associated peptide, CLIP (50).
Three lymphocyte classes (CD4 and
CD8 T cells, and NK cells) are important for resistance to
CMV during acute infection. CD8 T cells promote
MCMV clearance and mediate protective immunity (9),
and are also important for control of HCMV (53). As a
countermeasure, both MCMV and HCMV inhibit antigen
presentation to CD8 T cells by limiting MHC class I protein-dependent antigen presentation (12). HCMV and
MCMV prevent decreased expression of MHC class I on
infected cells from triggering NK cells via expression of cell
surface MHC class I homologs (17, 18). CD4 T cells and
IFN- are important for clearance of virus from the salivary
gland (1, 11), but priming of CD4 T cells is inhibited in
MCMV-infected mice (19). The findings in this paper delineate a potential mechanism by which MCMV might impair CD4 T cell activation and avoid recognition by this
third important class of lymphocytes.
We believe that viral impairment of IFN--induced antigen presentation by M
s in particular has important implications for acute and chronic MCMV pathogenesis. M
s
are involved in dissemination of MCMV and HCMV (25-
27) and yet they are activated by IFN-
during acute
MCMV infection (3, 23). IFN-
inhibits MCMV growth
in M
s and other cells (3, 54, 55). How then does the virus
disseminate in a cell activated by an anti-viral cytokine? We
propose that signaling by IFN-
is inhibited in M
s if the virus is able to infect them and express protein before activation by IFN-
. This would allow the virus to take advantage of the mobility of the M
s for dissemination,
while minimizing the effects of IFN-
on viral growth and
enhancement of immune induction. Our hypothesis is supported by the observation that M
-like cells that disseminate MCMV are MHC class II negative (25), whereas the
majority of M
s responding to MCMV in inflammatory
exudates from immunocompetent mice express high levels
of MHC class II and are uninfected (3, 23). Furthermore,
we have preliminary data for a role of IFN-
in controlling
chronic MCMV infection (Presti, R., J. Pollock, and H.W.
Virgin, unpublished data). Since one site of MCMV and
HCMV latency is cells of the monocyte/M
lineage, interference with IFN-
signaling in latently infected cells may
permit viral escape from IFN-
's antiviral effects, allowing
reactivation and subsequent growth.
We found that MCMV infection inhibits expression of specific IFN--induced genes. Other viruses
such as adenovirus have also been shown to alter IFN-mediated gene induction. Adenovirus protein E1A may block
IFN-
signaling by inhibition of STAT2-dependent transactivation involving p300/CBP (56). In our studies,
MCMV inhibited IFN-
signaling distal to STAT1
activation since induction of STAT1
DNA binding activity
was normal in infected cells, and mRNA levels of IRF-1, a
gene transactivated by STAT1
were also near normal
early after IFN-
treatment. Constitutive and inducible expression of MHC class II, H-2Ma, H-2Mb, and Ii are dependent on the class II transactivator (CIITA) (57).
MCMV significantly inhibits induced expression of all of
these genes. Therefore, one may speculate that a mechanism of viral action is the specific impairment of expression
or function of CIITA.
Although there is a formal possibility that MCMV exerts
its effect at the level of mRNA stability, we doubt this for
two reasons. First, we saw little evidence of a general effect
of MCMV on mRNA stability in the presence or absence
of IFN- over multiple experiments based on comparisons
of cyclophilin,
-actin, and CD45 mRNA levels. Second,
the effects of MCMV are specific to certain MHC genes. Thus, if MCMV infection interferes with IFN-
induction
of MHC genes by destabilizing mRNAs, one would have
to argue that the effects are specific for certain transcripts
(e.g., IAb and Ii but not TAP2,
-actin, or cyclophilin).
We have documented a novel mechanism of immune
avoidance by CMV, selective blockade of IFN- induction
of genes involved in antigen presentation to CD4 T cells.
Further characterization of MCMV's block on IFN-
signaling, and identification of the viral genes involved will
likely enhance understanding of CMV pathogenesis and
the antiviral functions of the immune system.
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Footnotes |
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Address correspondence to Herbert W. Virgin IV, Department of Pathology, Box 8118, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Phone: 314-362-9223; Fax: 314-362-4096; E-mail: virgin{at}immunology.wustl.edu
Received for publication 12 December 1997 and in revised form 5 February 1998.
We would like to acknowledge helpful discussions that occurred during lab meetings shared with Dr. Sam Speck and Dr. David Leib. We would like to give special thanks to Dr. Robert Schreiber; Dr. Erika Bach; Dr. Keith Pinkard; Scott Rodig; Joan Riley and Dale Campbell, who supplied both advice and critical reagents for analysis of STAT1 activation; Dr. Paul Allen who supplied the T cell hybridoma and peptide antigen used in antigen presentation studies; Dr. John Monaco who supplied several mouse cDNAs; and Dr. Edward Mocarski, who supplied MCMV mutant RM427.This work was supported by a grant to H.W. Virgin IV from the National Institute of Allergy and Infectious Diseases (RO1 AI-39616). H.W. Virgin was additionally supported by the Monsanto/Searle Biomedical Agreement. M. Connick was supported by National Institutes of Health (NIH) training grant AI-01763. M.T. Heise was supported by NIH training grant ST32 AI-07163.
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