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J. Exp. Med., Volume 187, Number 7, April 6, 1998 1037-1046

Murine Cytomegalovirus Inhibits Interferon gamma -induced Antigen Presentation to CD4 T Cells by Macrophages Via Regulation of Expression of Major Histocompatibility Complex Class II-associated Genes

By Mark T. Heise, Megan Connick, and Herbert W. Virgin IV

From the Center for Immunology and Departments of Pathology and Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110

    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CD4 T cells and interferon gamma  (IFN-gamma ) 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-gamma induced antigen presentation to CD4 T cells. MCMV infection inhibited IFN-gamma -induced presentation of major histocompatibility complex (MHC) class II associated peptide antigen by differentiated bone marrow macrophages (BMMphi s) to a T cell hybridoma via impairment of MHC class II cell surface expression. This effect was independent of IFN-alpha /beta induction by MCMV infection, and required direct infection of the BMMphi s with live virus. Inhibition of MHC class II cell surface expression was associated with a six- to eightfold reduction in IFN-gamma induced IAb mRNA levels, and comparable decreases in IFN-gamma induced expression of invariant chain (Ii), H-2Ma, and H-2Mb mRNAs. Steady state levels of several constitutive host mRNAs, including beta -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-gamma -induced transporter associated with antigen presentation (TAP)2 mRNA levels were minimally altered in infected cells. Analysis of early upstream events in the IFN-gamma signaling pathway revealed that MCMV did not affect activation and nuclear translocation of STAT1alpha , and had minor effects on the early induction of IRF-1 mRNA and protein. We conclude that MCMV infection interferes with IFN-gamma -mediated induction of specific MHC genes and the Ii at a stage subsequent to STAT1alpha 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.

    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-alpha /beta , TNF-alpha , IL-12, and IFN-gamma 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-gamma (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-gamma (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-beta , as well as direct infection, inhibits IFN-gamma -induced MHC class II expression in endothelial cells (20, 21). HCMV also alters MHC class II expression in cultured human peripheral blood macrophages (Mphi s; reference 22). Similarly, MCMV-induced IFN-alpha /beta inhibits MHC class II expression on Mphi s during the innate immune response (23).

Mphi s are important in the pathogenesis of both HCMV and MCMV. Key functions of Mphi s include presentation of antigen to CD4 T cells via MHC class II and secretion of cytokines. Mphi 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 Mphi s or Mphi -like cells within the blood (25). Monocytes and Mphi s are a site of long-term latency for HCMV and MCMV (28).

In these studies, MCMV impaired IFN-gamma -induced MHC class II-dependent antigen presentation by bone marrow (BM)Mphi s. This effect was due to the failure of IFN-gamma 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.

    Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals, Media, and BMMphi Culture.

4-12-wk-old 129Ev/Sv and IFNalpha beta receptor-deficient (IFNR-alpha /beta R-/-) mice (32) were housed in a Biosafety Level 2 facility at Washington University in accordance with all Federal and University policies. Sentinel animals were negative for adventitious mouse pathogens by serology. Virus stocks were grown and diluted in low endotoxin (<0.025 ng/ml) DME containing 10% FCS (DME 10%; reference 3). BMMphi s were prepared as previously described (23) and were at least 95% F4/80 positive (data not shown). BMMphi cultures were mock infected or infected with MCMV or UV-inactivated MCMV, at a multiplicity of infection (MOI) of 5.0 unless otherwise stated, for 1 h at 37°C in 5% CO2 with rocking every 5 min. After infection, cells were treated with IFN-gamma (100 IU/ml; Genentech, San Francisco, CA) or medium alone for various periods of time while incubating at 37°C in 5% CO2. Cells were harvested by scraping, then fixed, and analyzed for IAb expression by flow cytometry. BMMphi viability was determined by trypan blue exclusion.

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 bacterial beta -galactosidase (RM427; reference 25) was a gift of Dr. Edward Mocarski (Stanford University, Stanford, CA). 3T12 cells as well as MCMV stocks were negative for mycoplasma using the Mycoplasma TC test kit (Gen-Probe, San Diego, CA).

Analysis 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 BMMphi were separated by fluorescent activated cell sorting on a FACS® Vantage fluorescent activated cell sorter (Becton Dickinson, San Jose, CA). beta -galactosidase expression in sorted cells was detected by 5-bromo-4-chloro-3 indolyl-beta -D-galactopyranoside staining (33).

Supernatant Transfer Studies.

Wild-type 129 BMMphi s were either mock infected or infected with MCMV for 48 h. Supernatants were ultracentrifuged for 30 min at 100,000 g to remove free virus as confirmed by plaque assay, and then placed on naive cultures of wild-type or IFN-alpha /beta R-/- BMMphi s at 2 ml/plate in 60-mm dishes (Sarstedt, Newton, NC) for 1 h. In additional groups, MCMV was added to the cultures at an MOI of 5.0. After 1 h of incubation, IFN-gamma (100 IU/ml) was added and culture volumes were raised to 3 ml with fresh medium. Cultures were incubated at 37°C for 48 h and were assayed for IAb expression.

Electrophoretic Mobility Shift Assays.

BMMphi s were either mock infected or infected with MCMV for 1 h at 37°C. For STAT1alpha activation, cells were harvested at 1, 24, and 48 h after infection. 106 cells were suspended in endotoxin free PBS (Sigma Chemical Co., St. Louis, MO) containing 10% FCS, and were treated with IFN-gamma at concentrations ranging from 0.5 to 1,000 IU/ml for 10 min. Nuclear extracts were prepared and assayed by electrophoretic mobility shift assay (EMSA) against a 32P-labeled oligonucleotide probe derived from the IFN-gamma activating sequence of the Fcgamma RI promoter (34). Anti-STAT1 super-shift assays were performed by incubation of 2 µg of STAT1 (p91) specific antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) with the nuclear extract/ probe mixture for 45 min at 25°C before acrylamide gel electrophoresis. For analysis of IRF-1 DNA-binding activity, BMMphi were removed from medium containing L cell conditional medium (23) for 15 h before infection with MCMV for 1 h followed by treatment with IFN-gamma (100 IU/ml) for an additional 4 h. Nuclear extracts were generated from 106 cells (34) and assayed for IRF-1 DNA binding activity by EMSA using a 32P-labeled oligonucleotide containing the IFN response factor (IRF)-E of the murine (2'-5') oligoadenylate synthase promoter (35). Super-shift assays for IRF-1 were performed by adding 2 µg of anti-murine IRF-1 polyclonal antisera (Santa Cruz Biotechnology) to mixtures of extract and probe and incubating at 25°C for 45 min before analysis.

Northern Blot Analysis.

Total cellular RNA was harvested from BMMphi cultures using RNAzol (Tel-Test Inc., Friendswood, TX) and analyzed by Northern blot hybridization (36). PCR-derived probes used for these studies included a 300-bp fragment of the murine IAb beta chain (5'-ccggaattcgccagtgcctccagaggtgacagtgtatc; 3'-cgcggatccccatgccacagaaacaggtctcaggag), a rat beta -actin fragment (5'-tatggagaagatttggcacc; 3'-gtccagacgcaggatggcat; gift of Dr. J. Milbrandt, Washington University) and a 226-bp fragment of mouse Ii p33 cDNA (5'-agctctgtacaccggtgtctctgtcc; 3'-gacattggacgcatcagcaagggag; gift of Dr. E. Unanue, Washington University, St. Louis, MO). The following cDNAs were also used to generate probes: murine IRF-1 (37), rat cyclophilin (38), murine CD45 (provided by Dr. M. Thomas, Washington University), TAP1 (39), TAP2 (40), H-2Ma, and H-2Mb (41) (the latter four gifts of Dr. J. Monaco, University of Cincinnati, Cincinnati, OH). All probes were radiolabeled using the Megaprime DNA Labeling System (Amersham Corp., Arlington Heights, IL). Loading of mRNA was normalized against 28S ribosomal RNA (42) or against cyclophilin or beta -actin mRNA levels. Northern blot hybridizations were quantitated on a Phosphorimager (Molecular Dynamics, Sunnyvale, CA). Fold reduction of IFN-gamma -induced mRNA levels by MCMV was computed as [(IFN-gamma -stimulated signal in uninfected cells) - (unstimulated signal in uninfected cells)]/[(IFN-gamma -stimulated signal in infected cells) - (unstimulated signal in infected cells)], after signal was normalized to 28S rRNA or cyclophilin levels.

Antigen Presentation Assays.

BMMphi s were mock infected or infected with MCMV and treated with IFN-gamma (100 IU/ml) for 24 h. Cells were then harvested, counted, and incubated in 1% paraformeldahyde in RPMI 10% FCS for 15 min at 25°C. The fixed cells were washed vigorously, plated at 103, 5 × 103, 104, or 5 x 104 cells/well, and incubated with the T cell hybridoma B11 (IAb-restricted, beta -galactosidase peptide 429-441-specific, provided by Dr. Paul Allen, Washington University) in the presence of beta -galactosidase peptide 429-441, control peptide, or medium alone for 24 h. Supernatants were then assayed for IL-2 by proliferation of the IL-2-dependent cell line CTLL2 as measured by [3H]thymidine incorporation (43). No IL-2 production above background was observed using a control peptide derived from the MCMV MCK-1 open reading frame (VVLVVSTVADLREPC; reference 44) at 10, 1, or 0.1 µM (data not shown).

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MCMV Inhibition of Antigen Presentation to CD4 T Cells.

We examined IFN-gamma -stimulated presentation of peptide antigen to CD4 T cells by primary BMMphi 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). Mphi s were either mock infected or infected with MCMV, treated with IFN-gamma for 24 h, fixed, and tested for the ability to present peptide antigen (Fig. 1 A). IFN-gamma treatment of Mphi s resulted in effective presentation of beta -galactosidase peptide (429-441) to the T cell hybridoma B11, whereas MCMV infection abolished IFN-gamma - induced antigen presentation. The antigen presenting cell, rather than the T cell hybridoma, was impaired by virus since (a) Mphi s were fixed before incubation with T cells, and (b) addition of fixed MCMV infected Mphi s did not efficiently inhibit the capacity of uninfected Mphi 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 Mphi s failed to respond to IFN-gamma stimulation by upregulation of cell surface IAb (Fig. 1 B).


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  		Fig. 1.   MCMV infection impairs IFN-gamma -enhanced peptide presentation by MHC class II. 129 mouse BMMphi s were mock infected or infected with MCMV at an MOI of 5.0 for 1 h, and stimulated with IFN-gamma (100 IU/ml) or medium alone. After 24 h BMMphi s were assayed for ability to present peptide antigen, and analyzed for MHC class II expression by flow cytometry. (A) Mphi s were plated at 103, 5 × 103, 104, and 5 × 104 cells per well, and incubated with the T cell hybridoma B11 (beta -galactosidase 429-441, IAb-restricted) in the presence of beta -galactosidase peptide (429-441) at a concentration of 10 µM. Supernatants were harvested and assayed for IL-2 content using [3H]thymidine incorporation by CTLL2 cells. Mock + IFN-gamma and MCMV + IFN-gamma BMMphi s were mixed 1:1 and assayed as a control for potential toxic effects of MCMV-infected cells. Similar data was obtained at a peptide concentration of 1.0 µM. Data is from one of two similar experiments. All groups were plated in triplicate and are shown as mean cpm ± SEM. (B) Cell surface expression of the MHC class II molecule IAb on BMMphi s infected with MCMV and stimulated with IFN-gamma as above was measured by flow cytometry.

MCMV Blockade of IFN-gamma Induction of IAb Cell Surface Expression.

MCMV and HCMV infection at a low MOI impair IFN-gamma -induced MHC class II expression via induction of IFN-alpha /beta (20, 23). To evaluate MCMV's effects on Mphi responsiveness to IFN-gamma independently of IFN-alpha /beta , all experiments were performed at a high MOI in Mphi s derived from mice carrying a null mutation in the IFN-alpha /beta receptor (IFN-alpha /beta R-/-; reference 32). These Mphi s do not respond to IFN-alpha /beta stimulation, but do respond to stimulation with IFN-gamma as measured by induction of IAb expression (Fig. 1 B and Fig. 2; references 23, 32). Infection of IFN-alpha /beta R-/- Mphi s with increasing doses of MCMV resulted in a progressive decrease in the percentage of cells expressing IFN-gamma -induced IAb (Fig. 2). Cell viability in the MCMV-infected Mphi 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 Mphi IAb expression (Fig. 2).


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  		Fig. 2.   Infectious MCMV impairs IFN-gamma -induced MHC class II expression on Mphi s in a dose-dependent manner. IFN-alpha /beta R-/- BMMphi s were mock infected or infected with live or UV-inactivated MCMV at an MOI of 1.5, 3.0, or 6.0 for 1 h before addition of 100 IU/ml IFN-gamma . Mphi s were harvested 48 h after infection and assayed for MHC class II expression (IAb) by flow cytometry. Shown is one of three experiments, each of which yielded similar results.

Inhibition of MHC Class II Induction Correlates with Direct Infection of Mphi s.

MCMV-mediated inhibition of IFN-gamma - induced IAb expression was dependent on viral dose (Fig. 2), which suggested a role for direct infection. To address this possibility, IFN-alpha /beta R-/- Mphi s were infected with RM427 (25), a recombinant MCMV expressing beta -galactosidase under the MCMV ie1/ie2 promoter/enhancer at an MOI of 0.6, and treated with IFN-gamma . 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 Mphi s were enriched for beta -galatosidase positive (infected) cells (Fig. 3, A and B), whereas the IAb high cells were only 0.5-2.5% positive for beta -galactosidase. This demonstrated that IAb expression is low in MCMV-infected BMMphi s. However, over several experiments, beta -galactosidase was detected in <100% of IAb low cells, raising the possibility that a soluble mediator might contribute to low class II expression in beta -galactosidase-negative cells.


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  		Fig. 3.   MCMV-infected BMMphi s express low levels of MHC class II. IFN-alpha /beta R-/- BMMphi s were either mock infected or infected with recombinant MCMV expressing beta -galactosidase (RM427) at an MOI of 0.6. 1 h after infection, cells were treated with IFN-gamma (100 IU/ml) or medium alone for 48 h. Cells were harvested, stained for MHC class II (IAb) expression, and separated into MHC class II high and low populations by fluorescent activated cell sorting. The numbers of cells expressing beta -galactosidase in total and sorted cell populations was determined by 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside (x-gal) staining. (A) Mock-infected or infected cells (top panels), and IAb high and low populations from infected cultures (bottom panels) were stained for IAb. The dotted line represents staining on unstimulated, mock-infected cells. The solid line depicts staining of IFN-gamma - treated groups. (B) x-gal staining of mock- or MCMV-infected, and MHC class II high and low, populations is shown from one of three similar experiments.

To determine whether soluble factors played a role in MCMV's inhibition of IAb induction in IFN-alpha /beta R-/- Mphi s, supernatant transfer studies were performed. Supernatants from wild-type 129 Mphi s infected with MCMV or mock infected for 48 h were ultracentrifuged to remove free virus and placed on naive Mphi cultures derived from either wild-type 129 or IFN-alpha /beta R-/- mice. IFN-gamma 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 Mphi s inhibited IFN-gamma -induced IAb expression on wild-type Mphi s, but not on IFN-alpha /beta R-/- Mphi s (Fig. 4), confirming that MCMV-induced IFN-alpha /beta can inhibit IFN-gamma -induced MHC class II expression. However, there was no evidence for a soluble mediator affecting IAb expression in MCMV-infected IFN-alpha /beta R-/- Mphi cultures (Fig. 4). Therefore, direct infection of the Mphi , rather than a soluble mediator, is likely to explain MCMV's effects on MHC IAb expression.


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  		Fig. 4.   Supernatants from MCMV-infected Mphi s inhibit IFN-gamma induction of MHC class II on normal but not IFN-alpha /beta R-/- BMMphi s. Wild-type 129 BMMphi s were either mock infected or infected with MCMV at an MOI of 5.0. 48 h after infection, supernatants were removed, ultracentrifuged to remove free virus, and placed on naive cultures of either 129 or IFN-alpha /beta R-/- BMMphi s. At the time of supernatant transfer, additional cultures were infected with MCMV (MOI = 5.0) as positive controls. After 1 h of incubation with the supernatants or MCMV, cells were treated with IFN-gamma (100 IU/ml), incubated for 48 h, and harvested. MHC class II (IAb) expression was determined by flow cytometry. Shown is data from one of two experiments yielding similar results. (US, unstained).

MCMV Inhibits IFN-gamma -induced MHC Class II and Invariant Chain mRNA Expression.

IFN-gamma 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-gamma -induced MHC class II gene, IAb. Infection with MCMV, but not UV-inactivated MCMV, decreased IFN-gamma -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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  		Fig. 5.   MCMV infection reduces IFN-gamma -induced MHC class II and Ii mRNA levels. IFN-alpha /beta R-/- BMMphi s were either mock infected or infected with MCMV or UV-inactivated MCMV at an MOI of 5.0. 1 h after infection, cells were treated with medium alone or with IFN-gamma (100 IU/ml). Total cellular RNA was harvested at either 24 or 48 h after infection and analyzed by Northern blot hybridization for the IAb beta chain or Ii. beta -actin hybridization is also shown. Similar results were obtained in three separate experiments.

Although a 6-8-fold reduction of IFN-gamma -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-gamma -inducible gene, Ii, which is essential for efficient cell surface expression of MHC class II (46, 47). Northern blot hybridization showed that IFN-gamma -induced Ii transcript levels are reduced 5->10-fold at 24 h and >10-fold at 48 h in MCMV-infected BMMphi cultures. The combined effect of MCMV infection on IFN-gamma -induced IAb and Ii mRNA accumulation plausibly explains most or all of the decreased MHC class II expression on MCMV-infected Mphi 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 beta -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 BMMphi s with actinomycin D for 5-10 h revealed diminished beta -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-gamma -induced IAb and Ii mRNA accumulation is not solely due to a general effect on mRNA levels by MCMV.


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  		Fig. 8.   MCMV infection inhibits H-2Ma, H-2Mb, and TAP1 mRNA expression, but not IFN-gamma -induced TAP2 mRNA expression. IFN-alpha / beta R-/- BMMphi were either mock infected or infected with MCMV or UV-inactivated MCMV at an MOI of 5.0 in the presence or absence of IFN-gamma (100 IU/ml) for 24 h. Total cellular RNA was harvested and analyzed by Northern blot. Blots were probed using radiolabeled probes for murine TAP1, TAP2, H-2Ma, H-2Mb, cyclophilin, and 28S ribosomal RNA. Shown is one of three experiments which yielded similar results.

MCMV Does Not Alter IFN-gamma Induction of STAT1alpha DNA Binding Activity.

IFN-gamma 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-gamma receptor signaling, activation and nuclear translocation of the latent cytoplasmic transcription factor STAT1alpha . Activation of STAT1alpha is essential for IFN-gamma induction of MHC class II expression (48). Mphi s were either mock infected or infected with MCMV for 1, 24, or 48 h before stimulation with IFN-gamma , preparation of nuclear extracts, and evaluation of STAT1alpha binding activity by EMSA (Fig. 6). The presence of STAT1alpha in the activated complex was confirmed by super-shift with antibody against STAT1alpha (Fig. 6), whereas antibody to IRF-1 did not result in retardation of the complex (data not shown). IFN-gamma treatment resulted in comparable levels of STAT1alpha DNA binding activity in both mock- and MCMV-infected Mphi s, at 1, 24 (Fig. 6), and 48 (data not shown) h after infection. STAT1alpha activation was also equivalent in mock- and MCMV-infected Mphi s after treatment with IFN-gamma at doses as low as 0.5 IU/ml (data not shown). We conclude that the IFN-gamma signaling pathway remains intact through STAT1alpha activation in MCMV-infected BMMphi s.


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  		Fig. 6.   STAT1alpha activation and nuclear translocation after IFN-gamma stimulation is normal in MCMV-infected Mphi s. IFN-alpha / beta R-/- BMMphi s were infected with MCMV at an MOI of 5.0 or mock infected for 1 or 24 h. Harvested cells were incubated with IFN-gamma for 10 min and nuclear extracts were prepared. Extracts were assayed for STAT1alpha activation by EMSA against a 32P-labeled IFN-gamma activation sequence containing oligonucleotide derived from the murine Fcgamma R1 promoter. The presence of STAT1alpha in the complex was determined by antibody super-shift using STAT1alpha -specific antiserum. Shown are STAT1alpha activation by IFN-gamma at 1 h (100 IU/ml IFN-gamma ) or 24 h (5 IU/ml IFN-gamma ) after infection. Similar results were obtained in four separate experiments.

MCMV Effects on IFN-gamma Induction of IRF-1.

To directly assess the effects of MCMV on the transactivating function of STAT1, we examined IFN-gamma induction of IRF-1 mRNA and protein. IRF-1 is rapidly induced at the transcriptional level after IFN-gamma stimulation in a STAT1alpha -dependent manner (48), with maximal mRNA expression by 2 h of cytokine treatment (49). Mphi s were mock infected or infected with MCMV at an MOI of 5.0 for 3 h and treated with IFN-gamma (100 IU/ml) for the final 2 h of infection. Northern blot analysis showed that induction of IRF-1 mRNA accumulation by IFN-gamma in MCMV-infected Mphi 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 Mphi s that were mock or MCMV infected for a total of 5 h, with IFN-gamma 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-gamma 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-STAT1alpha , antiserum. IFN-gamma -induced IRF-1 DNA binding activity was decreased by at most 50% in MCMV-infected Mphi s compared to mock-infected Mphi s. IRF-1 DNA binding activity could also be induced by IFN-gamma in Mphi s that had been infected with MCMV for 24 h (data not shown).


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  		Fig. 7.   IRF-1 mRNA and DNA binding activity is inducible by IFN-gamma in MCMV-infected Mphi s. (A) IFN-alpha /beta R-/- were either mock infected or infected with MCMV at an MOI of 5.0 for 3 h. Cultures were treated with IFN-gamma for the final 2 h of infection and assayed for IRF-1 mRNA levels by Northern blot hybridization. Shown is one of two experiments yielding similar results. (B) IFN-alpha /beta R-/- BMMphi s were infected with MCMV at an MOI of 5.0 or mock infected for 5 h. Cells were treated with IFN-gamma for the final 4 h of infection and nuclear extracts were prepared. IRF-1 DNA binding activity was assessed by incubating extract with a 32P-labeled IRF-E oligonucleotide probe, which was then analyzed by EMSA. The presence of IRF-1 within the complex was confirmed by incubation with antiserum against murine IRF-1 or STAT1alpha . Three separate experiments yielded similar results.

MCMV Inhibits IFN-gamma Induction of Specific MHC Genes.

To assess the specificity of the block of IFN-gamma signaling caused by MCMV infection, we examined levels of several IFN-gamma -induced transcripts in addition to IAb, Ii, and IRF-1. IFN-alpha /beta R-/- Mphi s were infected with MCMV or UV-inactivated virus, or mock infected for 24 or 48 h in the presence or absence of IFN-gamma , and mRNA levels were analyzed by Northern blot hybridization. IFN-gamma induction of H-2Ma and H-2Mb expression was significantly inhibited by MCMV at 24 h (Fig. 8), whereas IFN-gamma -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-gamma induction of TAP2 transcript levels by MCMV demonstrated that MCMV selectively affects expression of a subset of IFN-gamma - inducible genes.

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-gamma induction of IAb and Ii gene expression in IFN-alpha /beta R-/- BMMphi s, resulting in defective antigen presentation to CD4 T cells. Inhibition of IAb expression required direct infection of the Mphi s with live virus. Early IFN-gamma signaling events including STAT1alpha 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-gamma -inducible MHC gene was largely unaffected.

Effects of MCMV on MHC Class II Antigen Presentation.

MCMV impaired the ability of IFN-gamma 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-gamma -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-gamma 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).

Implications of beta -herpesvirus Impairment of Antigen Presentation to CD4 T Cells.

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-gamma 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-gamma -induced antigen presentation by Mphi s in particular has important implications for acute and chronic MCMV pathogenesis. Mphi s are involved in dissemination of MCMV and HCMV (25- 27) and yet they are activated by IFN-gamma during acute MCMV infection (3, 23). IFN-gamma inhibits MCMV growth in Mphi 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-gamma is inhibited in Mphi s if the virus is able to infect them and express protein before activation by IFN-gamma . This would allow the virus to take advantage of the mobility of the Mphi s for dissemination, while minimizing the effects of IFN-gamma on viral growth and enhancement of immune induction. Our hypothesis is supported by the observation that Mphi -like cells that disseminate MCMV are MHC class II negative (25), whereas the majority of Mphi 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-gamma 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/Mphi lineage, interference with IFN-gamma signaling in latently infected cells may permit viral escape from IFN-gamma 's antiviral effects, allowing reactivation and subsequent growth.

Mechanism of Inhibition of IFN-gamma Induction of MHC Gene Expression.

We found that MCMV infection inhibits expression of specific IFN-gamma -induced genes. Other viruses such as adenovirus have also been shown to alter IFN-mediated gene induction. Adenovirus protein E1A may block IFN-alpha signaling by inhibition of STAT2-dependent transactivation involving p300/CBP (56). In our studies, MCMV inhibited IFN-gamma signaling distal to STAT1alpha activation since induction of STAT1alpha DNA binding activity was normal in infected cells, and mRNA levels of IRF-1, a gene transactivated by STAT1alpha were also near normal early after IFN-gamma 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-gamma over multiple experiments based on comparisons of cyclophilin, beta -actin, and CD45 mRNA levels. Second, the effects of MCMV are specific to certain MHC genes. Thus, if MCMV infection interferes with IFN-gamma 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, beta -actin, or cyclophilin).

We have documented a novel mechanism of immune avoidance by CMV, selective blockade of IFN-gamma induction of genes involved in antigen presentation to CD4 T cells. Further characterization of MCMV's block on IFN-gamma signaling, and identification of the viral genes involved will likely enhance understanding of CMV pathogenesis and the antiviral functions of the immune system.

    Footnotes

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.
1   Abbreviations used in this paper: BMMphi , bone marrow macrophage; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay; HCMV, human CMV; IRF, IFN response factor; Mphi , macrophage; MCMV, murine CMV; MOI, multiplicity of infection; TAP, transporter associated with antigen presentation.

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.

    References
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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