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The Journal of Immunology, 2002, 169: 167-176.
Copyright © 2002 by The American Association of Immunologists

Human Cytomegalovirus Disrupts Constitutive MHC Class II Expression1

Colleen M. Cebulla*, Daniel M. Miller{ddagger}, Yingxue Zhang*, Brian M. Rahill*, Peter Zimmerman*, John M. Robinson{dagger} and Daniel D. Sedmak2,*

Departments of * Pathology, and {dagger} Physiology and Cell Biology, Ohio State University College of Medicine and Public Health, Columbus, OH 43210; {ddagger} Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, FL 33136 Workshop. C.M.C. was a Presidential Fellow at Ohio State University. D.M.M. was a Howard Hughes Medical Institute Predoctoral Fellow.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD8+ and CD4+ T lymphocytes are important in controlling human CMV (HCMV) infection, but the virus has evolved protean mechanisms to inhibit MHC-based Ag presentation and escape T lymphocyte immunosurveillance. Herein, the interaction of HCMV with the MHC class II Ag presentation pathway was investigated in cells stably transfected with class II transactivator. Flow cytometry experiments demonstrate that HCMV infection decreases cell-surface MHC class II expression. HCMV down-regulates MHC class II surface expression without a significant effect on class II RNA or steady-state protein levels. SDS-stability and confocal microscopy experiments demonstrate normal levels of steady-state peptide-loaded class II molecules in infected cells and that class II molecules reach late endosomal and HLA-DM positive peptide-loading compartments. However, MHC class II positive vesicles are retained in an abnormal perinuclear distribution. Finally, experiments with a mutant HCMV strain demonstrate that this novel mechanism of decreased MHC class II expression is not mediated by one of the known HCMV immunomodulatory genes. These defects in MHC class II expression combined with previously identified CMV strategies for decreasing MHC class I expression enables infected cells to evade T lymphocyte immunosurveillance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cytomegalovirus is an important cause of morbidity and mortality in immunocompromised populations, e.g., AIDS patients and transplant recipients (1). Human CMV (HCMV),3 like other HSVs, is able to persist for the life of the host, even in immunocompetent individuals. Reactivation of latent or persistent virus is responsible for the majority of HCMV-associated morbidity and mortality in immunosuppressed hosts. Thus, determining the factors that promote HCMV persistence is a critical step in understanding and preventing HCMV disease.

Cell-mediated immunity is essential in controlling HCMV and other viral infections (2, 3, 4, 5, 6, 7). However, viruses have evolved remarkable strategies for evading cell-mediated immune responses (8). Moreover, a single virus can use multiple means of blocking Ag presentation. For example, the HCMV US2, US3, US6, and US11 gene products use distinct mechanisms to inhibit MHC class I Ag presentation and escape CD8+ T cell responses (9, 10, 11, 12, 13, 14, 15, 16). In addition, the HCMV matrix protein pp65 inhibits the presentation of HCMV IE1 protein-derived peptides to IE1-specific CD8+ T cells (17).

It is well-established that CD4+ T cells are important in controlling CMV infection. Mice depleted of CD8+ T cells halt disseminated CMV disease with similar kinetics as nondepleted controls, suggesting a compensatory antiviral role of the CD4+ population (18). In addition, clearance of CMV from the salivary gland, an organ important for persistence, is dependent on CD4+ T cells (19, 20). Control of CMV replication in the salivary gland is dependent on CD4+ T cells with the TH-1 phenotype and IFN-{gamma} is an essential factor (20). CMV-specific CD4+ T cells play a critical role in controlling CMV infection through the release of IFN-{gamma}, but also have been shown to mediate cytolysis of infected cells in an MHC class II-restricted manner (20, 21, 22, 23).

CD4+ T cells recognize viral protein-derived peptides in the context of MHC class II molecules. MHC class II proteins are constitutively expressed in APCs such as monocyte/macrophages, dendritic cells, and B cells. MHC class II {alpha}- and {beta}-chains form a heterodimer in the endoplasmic reticulum and associate with the invariant chain (Ii) to form a "nonameric" complex (24, 25, 26). This complex moves through endosomal compartments to peptide-loading compartments where the HLA-DM molecule facilitates the exchange of the class II-associated Ii peptide portion of Ii for peptides generated in lysosomal compartments (24, 26). Class II molecules present these peptides to CD4+ T cells on the cell surface.

CMV blocks IFN-{gamma}-stimulated MHC class II expression in endothelial cells, an important site of CMV infection in vivo (27, 28). HCMV inhibits IFN-{gamma} inducible MHC class II expression by blocking the JAK/STAT signal transduction pathway in infected cells and murine CMV blocks IFN-{gamma}-stimulated MHC class II expression through a JAK/STAT-independent mechanism (29, 30, 31).

HCMV infection decreases constitutive MHC class II expression in monocyte/macrophages, a critical site of HCMV latency and reactivation (32). There is evidence that the HCMV US2 glycoprotein, a molecule previously shown to target MHC class I H chains for degradation, mediates a decrease in HLA-DR{alpha} and HLA-DM expression at early times after infection (33). Given the fact that HCMV uses four distinct gene products to block MHC class I expression and that these gene products target distinct levels of the class I Ag processing pathway, we suspected additional, US2-independent mechanisms for the decrease in MHC class II expression in infected cells.

Herein, we investigate the HCMV-mediated disruption of constitutive MHC class II expression by generating a model system that facilitates molecular analyses. Monocyte/macrophages, which are an important site of HCMV infection in vivo and constitutively express MHC class II, are not efficiently infected in vitro, thereby limiting investigations of the molecular mechanism for decreased class II in these primary cells. To generate a model to study the interaction of HCMV with MHC class II, we transfected a class II negative, HCMV permissive cell line, U373 astrocytoma cells, with class II transactivator (CIITA) cDNA, thereby creating cells that constitutively express class II molecules. CIITA is the "master switch" in class II expression, and transfection of CIITA into class II negative cells results in constitutive MHC class II, Ii, and HLA-DM expression (34). U373 cells are permissive for HCMV infection and have been used for investigating HCMV/MHC class I interactions (10, 11, 12, 13, 33). Using this model system, we have discovered a mechanism for the HCMV-mediated disruption of MHC class II expression that is independent of the effects of US2 on MHC class II proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and transfections

U373/CII cells were generated by stable transfection of U373 cells (American Type Culture Collection, Manassas, VA) with full-length CIITA cDNA (a generous gift from Dr. J. Boss, Emory University School of Medicine, Atlanta, GA), driven by the CMV IE promoter, in vector pcDNA 3.1 using SuperFect reagent (Qiagen, Valencia, CA). Stable transfectants were selected by adding 400 µg/ml of G418 (Life Technologies, Grand Island, NY) to culture media (DMEM with 10% FBS). FACS sorting was performed on stably transfected cells and only cells with high levels of HLA-DR expression were collected. An individual clone, U373/CII, with high HLA-DR surface levels was isolated with cloning rings. Control cells, U373/pc, were generated by transfecting U373 cells with the pcDNA 3.1 vector with no insert and selecting for stable transfection with G418.

CMV infection and viral stock preparation

U373/CII were infected with Towne HCMV, AD169 HCMV, or the HCMV deletion mutant RV7186, which is deleted for IRS1-US11 (35) at a multiplicity of infection of 10. Infection levels were confirmed by HCMV IE immunofluorescence (clone MAB810; Chemicon International, Temecula, CA) and were 80–90%. For analysis of HCMV gene expression, U373/CII were infected in the presence of 300 µg/ml phosphonoformic acid (PFA), an inhibitor of HCMV late gene expression.

Cell-free viral stocks of HCMV were prepared as previously described (30). Briefly, virus was propagated in fibroblasts (MRC-5), passages 22–35, at low multiplicity of infection. Virions were stored frozen at -80°C and standard plaque assays were used to quantitate viral titers from representative vials.

RT-PCR

Total RNA isolated using guanidine thiocyanate extraction and cesium chloride centrifugation (36) was reverse transcribed into cDNA using the SuperScript Preamplification System (Life Technologies), and 500 ng cDNA was used per PCR. PCR (50 µl) were performed using PCR buffer (20 mM Tris-HCL (pH 8.4), 50 mM KCl), 1.5 mM MgCl2, 20 mM dNTP, 400 nM of each sense and antisense primer, and 5 U Taq polymerase (Life Technologies). PCR were incubated for 20 cycles on an Ericomp Easy Cycler (Ericomp, San Diego, CA). Previously published CIITA primers were used (37). The {beta}-actin primers used were sense (5'-GTGGGGCGCCCCAGGCACCA-3') and antisense (5'-CTCCTTAATGTCACGCACGATTTC-3'). PCR analysis of US2 and US12 was performed on phenol:chloroform-extracted DNA (36) isolated from viral stocks of Towne and RV7186. The primer sets used for US2 were sense (5'-ATGAACAATCTCTGGAAAGCC-3') and antisense (5'-TCAGCACACGAAAAACCGCAT-3'), and for US12 were sense (5'-CGGAATTCATGGTACAGATCCAGTTTGCAC-3') and antisense (5'-GCCCTAGGATATTTATGAAAAAGCCAGTGTGCC-3').

Northern blot analysis

Total RNA from U373/CII and U373/pc was extracted at 0, 1, and 3 days after infection using the guanidine thiocyanate extraction and cesium chloride centrifugation method (36). The RNA was separated on a 1.4% agarose 0.22-M formaldehyde gel. Northern blots were probed for HLA-DR{alpha} as well as the loading control GAPDH. Probes were generated using random priming and [{alpha}-32P]dCTP (Deca Prime kit; Ambion, Austin, TX). cDNA templates were generated for the HLA-DR{alpha} probes using primer sets sense (5'-AAAGCGCTCCAACTATACTCCGA-3') and antisense: (5'-ACCCTGCAGTCGTAAACGTCC-3') and clone p0A1 (ATCC) was used for the GAPDH probes. Dilutional standards of 10, 5, and 2.5 µg RNA isolated from noninfected and HCMV-infected U373/CII were run as controls. Densitometry analyses were performed by scanning autoradiographic films with a Hewlett Packard flatbed scanner (Hewlett Packard, Palo Alto, CA), and the digital images were analyzed using Scion Image software (Scion Corporation, Frederick, MD).

Flow cytometry

Cells were harvested with trypsin/EDTA, labeled with directly conjugated fluorescein-labeled Abs, and washed three times with SBSS. Abs for monomorphic HLA-DR epitopes (clone Tu36 from GenTrak (Plymouth Meeting, PA; Refs. 38, 39, 40); and L243 (41) from BD PharMingen, San Diego, CA) were used as well as MHC class I (anti-HLA-A, B, C; ICN Biomedicals, Irvine, CA). A minimum of 5 x 103 cells were analyzed on a Coulter flow cytometer (Coulter, Hialeah, FL) calibrated with 2% fluorescent microbeads (Standard Brite Beads; Coulter). Nonspecific fluorescence was assessed by staining cells with an isotype control Ab. Mean fluorescence intensity derived from a linear scale was reported by subtracting nonspecific mean fluorescence intensity values from MHC class I or class II mean fluorescence intensity values.

Immunoprecipitation, Western analysis, and SDS-stability assay

For HLA-DR{alpha} and Ii immunoprecipitations, U373/CII and U373/pc cells were treated with lysis buffer containing 1% Triton X-100, 0.15 M NaCl, 50 mM Tris (pH 8.0), 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM PMSF, 1 mM orthovanadate, and 5 µg/ml each of pepstatin, leupeptin, and aprotinin. Samples were sonicated and precleared with an isotypic control Ab and protein G-Sepharose. The cleared supernatants were incubated with primary Ab, TAL.1B5 (HLA-DR{alpha}; DAKO, Glostrup, Denmark) (42), LN2 (Ii C terminus; BD PharMingen) or Pin1.1 (Ii N terminus; a generous gift of Dr. P. Cresswell, Yale University School of Medicine, New Haven, CT) (43). Immune complexes were collected with protein G-Sepharose, pelleted by centrifugation, washed in lysis buffer, and resuspended in SDS-PAGE loading buffer containing 0.2% 2-ME (36). Samples were boiled 5 min, Sepharose beads pelleted, and precipitated proteins fractionated by 12% SDS-PAGE. Western blot analyses used the same lysis buffer as for immunoprecipitation experiments. Samples were diluted 1/1 in sample buffer, boiled, and loaded on 12% SDS-PAGE gels.

For SDS-stability experiments, a group of cells were treated with 50 mM NH4Cl, to inhibit peptide loading of MHC class II as a negative control (44). SDS-stability lysis buffer containing 0.5% Nonidet P-40, 0.15 M NaCl, 50 mM Tris (pH 8.0), and protease inhibitors was used. Samples were treated with 3% SDS for 1 h at 25°C under nonreducing conditions, then split in half and either heated to 100°C or maintained at room temperature for 5 min. Samples were then loaded on 12% SDS-PAGE gels, transferred to nitrocellulose, and blocked in 5% nonfat dry milk.

Immunoblotting was performed using Abs: TAL.1B5 (HLA-DR{alpha}, DAKO; Refs. 42 and 45), DK22 (code M704, recognizing {beta}-chain of HLA-DR, DP, and DQ; DAKO) (46, 47), TU36 (recognizing HLA-DR{alpha}{beta} complexes; Caltag Laboratories, Burlingame, CA) (38, 39, 40), LN2 (Ii C terminus, BD PharMingen), or Pin1.1 (Ii N terminus, a generous gift of Dr. P. Cresswell; Ref. 43), followed by anti-mouse IgG-HRP secondary Ab, or polyclonal rabbit anti-STAT-2 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-rabbit IgG-HRP secondary Ab (Santa Cruz Biotechnology). Blots were developed using ECL (Amersham Pharmacia Biotech, Piscataway, NJ) or West Dura Super Signal (Pierce, Rockford, IL) chemiluminescence detection systems. Densitometry analyses of Western and immunoprecipitation films were performed as for Northern blot autoradiographic films.

Immunofluorescence and confocal microscopy

Infected and noninfected U373/CII cells were grown on glass chamberslides (Nunc, Naperville, IL), then fixed using a modification of Muczynski et al. (48). Briefly, at 3 days after infection, cells were rinsed with PBS/30 mM sucrose, fixed for 10 min with 4% paraformaldehyde, and permeabilized with 0.2% saponin (in PBS/30 mM sucrose/1% BSA). Cells were stained as follows: anti-CMV IE Ab (clone MAB810, Chemicon International), lysosmal-associated membrane protein 1 for late endosomes/early lysosomes (clone BB6, a generous gift of Dr. M. Fukuda, La Jolla Cancer Research Center, Burnham Institute, La Jolla, CA; Ref. 49), HLA-DM (clone MaP.DM1, a generous gift of Dr. P. Cresswell; Ref. 50), Ii (clone Pin1.1, a generous gift of Dr. P. Cresswell; Ref. 43). These primary Abs were followed by a goat-anti-mouse F(ab')2 secondary Ab conjugated to Rhodamine Red-X (Jackson ImmunoResearch Laboratories, West Grove, PA). For dual labeling, cells were stained with an Ab recognizing HLA-DR, DP, and DQ isoforms (clone CR3/43 directly conjugated with FITC, DAKO; Ref. 51). Isotype controls were used for Abs CR3/43, and anti-CMV IE, and secondary Ab alone was used as a negative control for LAMP-1, HLA-DM, and Pin1.1. Results were analyzed on a Bio-Rad MRC 600 confocal microscope equipped with an argon-krypton laser (Bio-Rad, Hercules, CA). Excitation wavelengths were 488 and 568 nm, and detection wavelengths were 522 and 585 for FITC and rhodamine visualization, respectively. For dual localization, the images were merged using Confocal Assistant software (public domain software developed by T. C. Brelje, University of Minnesota, Minneapolis, MN).

Statistics

As appropriate, data are shown as the mean ± SEM. One-sided Student’s t tests were used to determine significant differences between groups (SigmaStat Statistical Analysis system; SPSS, Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cells that constitutively express MHC class II molecules (U373/CII) were generated by transfecting class II negative U373 cells with CIITA cDNA, FACS sorting for high MHC class II expression, and isolating an individual clone with cloning rings. Fig. 1GoA shows HLA-DR expression in the original CIITA-transfected population of U373 cells and high levels of HLA-DR expression in the isolated U373/CII clone (Fig. 1GoA). Stable transfection was confirmed by RT-PCR analysis (Fig. 1GoB).



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  		FIGURE 1. Generation of U373/CII cells. We generated a constitutive class II model system by transfecting U373 cells with a plasmid containing full-length CIITA cDNA (U373/CII cells) or with the vector alone pcDNA3.1 (U373/pc). A, left panels, Analysis of HLA-DR expression in U373 cells transfected with CIITA cDNA shows that ~75% of the cells are class II positive. A, right panel, FACS sorting selecting for high HLA-DR expression and isolation of a single transfected clone yields U373/CII cells which express HLA-DR. B, CIITA expression was verified in U373/CII cells and in a positive control cell line that constitutively expresses MHC class II molecules (Raji) by RT-PCR. U373/pc cells which were transfected with vector alone do not contain CIITA.

 
To analyze the effects of CMV infection on surface MHC class II expression, U373/CII were infected with the Towne strain of HCMV and surface HLA-DR expression was measured by flow cytometry. HCMV infection decreased HLA-DR levels by 2 days after infection (p = 0.01), with a maximal decrease at 3 days (p = 0.004), yielding a 60% reduction (±5%) in class II molecules on the surface of infected cells (Fig. 2GoA). Similar results were obtained using two anti-HLA-DR mAbs reactive to distinct portions of the HLA-DR molecule as well as infection with the AD169 CMV strain (data not shown).



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  		FIGURE 2. HCMV infection decreases constitutive MHC class II surface expression. U373/CII cells were infected with HCMV and cell surface MHC class II expression was analyzed by flow cytometry. A, HCMV infection results in decreased HLA-DR expression beginning 2 days after infection with the lowest levels detected at three days after infection. *, Statistically significant differences compared with the day 0 control (p = 0.01 for 2 days after infection, and p = 0.004 for the decrease 3 days after infection). B, HCMV infection decreases HLA-DR expression to levels analogous to the HCMV-mediated decrease in HLA-A, B, C (MHC class I) expression.

 
The CMV US2, US3, US6, and US11 glycoproteins mediate a decrease in MHC class I expression (9, 10, 11, 12, 13, 14, 15, 16). To confirm that a similar response occurs in U373/CII cells, we compared the steady-state levels of surface MHC class II expression to surface MHC class I expression in infected U373/CII cells. Both the kinetics of and absolute decrease in surface MHC class I molecules was similar to that observed for MHC class II molecules in CMV-infected U373/CII cells (Fig. 2GoB).

To test the hypothesis that a soluble factor is responsible for the decrease in constitutive class II surface expression, as it is in other viral infections (31, 52, 53), we performed supernatant transfer experiments (31). Supernatants from HCMV-infected U373/CII cells were cleared of infectious virions by centrifugation and transferred to noninfected U373/CII for 24 h at 37°C. Transfer of supernatants from HCMV-infected U373/CII cultures did not decrease class II surface expression on noninfected U373/CII cells (data not shown). Therefore, a direct cellular interaction between HCMV and U373/CII results in the decrease of MHC class II surface expression.

To explore the mechanism responsible for the decrease in MHC class II, we investigated steady-state class II mRNA and protein levels. Northern blot analyses of U373/CII cells demonstrate that there is no change in the relative levels of HLA-DR{alpha} mRNA at 3 days after HCMV infection, despite the significant decrease in surface HLA-DR expression at this time, suggesting that the decrease in class II expression is not due to alterations in steady-state MHC class II RNA (Fig. 3GoA). Dilutional Northern blot analyses of HCMV-infected and noninfected U373/CII 3 days after infection confirmed that differences in HLA-DR{alpha} mRNA levels in infected and noninfected cells were detectable with this assay (Fig. 3GoB). Analysis of HLA-DR{alpha} and HLA-DR{beta} protein expression by Western blot analysis at 3 days after infection demonstrates no change in steady-state levels of HLA-DR{beta} and only a slight decrease in HLA-DR{alpha} molecules (8%) by scanning densitometry compared with the levels of an internal control, STAT-2 with HCMV infection (Fig. 4Go). Furthermore, detection of steady-state HLA-DR{alpha} by immunoprecipitation followed by immunoblotting yields identical results.



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  		FIGURE 3. HCMV infection does not decrease HLA-DR{alpha} mRNA levels in U373/CII. A, Northern blot analysis of HLA-DR{alpha} RNA levels was performed with U373/CII at 3, 24, 48, and 72 h after infection. GAPDH was probed as a loading control. HCMV infection does not alter HLA-DR{alpha} RNA levels at 3 days after infection. B, Dilutional Northern analysis of noninfected and infected U373/CII HLA-DR{alpha} RNA levels at 3 days after infection was performed to confirm the detection range of the assay.

 


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  		FIGURE 4. Total cellular levels of MHC class II proteins are not significantly decreased by HCMV infection. A, Western blot analysis of total cell levels of HLA-DR{alpha} and HLA-DR{beta} at 3 days after infection in U373/CII was performed. There is no significant decrease in the steady-state levels of HLA-DR{beta} protein in HCMV-infected cells at 3 days after infection compared with the levels of an internal control STAT-2. B, There is only a slight decrease in steady-state HLA-DR{alpha} levels (8% as determined by denistomtery). Ab TAL.1B5 was used to detect HLA-DR{alpha} and DK22 used to detect HLA-DR{beta}.

 
Taken together, the data that class II RNA levels are unchanged in HCMV-infected U373/CII cells, and class II molecules are not significantly decreased, suggests that HCMV induces a defect in the MHC class II trafficking pathway. The Ii is a critical chaperone molecule for the proper trafficking of class II, and alterations in Ii production, cleavage, or phosphorylation can lead to altered trafficking of MHC class II (54, 55, 56, 57, 58, 59, 60). Immunoprecipitation experiments were performed to evaluate Ii levels in HCMV-infected cells. Whole-cell levels of p41, p35, and p33 Ii isoforms were comparable in infected and noninfected U373/CII cells as determined by immunoprecipitation with either a C-terminal (Fig. 5Go) or N-terminal-specific Ab (data not shown).



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  		FIGURE 5. Total cellular levels of Ii are not decreased by HCMV infection. Immunoprecipitation of Ii chain reveals that HCMV infection does not decrease the relative levels of the three Ii isoforms: p41, p35, and p33. This result was observed with Abs to the C terminus of Ii. Comparable results were obtained with an N terminus-binding Ii Ab. Equivalent proportion of lysate for the three lanes was verified by analyzing STAT-2 expression as an internal control (data not shown).

 
Although there is not a quantitative decrease of Ii in HCMV-infected cells, qualitative defects in Ii function may alter the quality of MHC class II molecules in infected cells, preventing the formation of mature, peptide-bound class II species (54, 56). To assess whether HCMV induces a defect in Ii function and to determine the maturation state of class II molecules in infected cells, we performed SDS-stability Western blot experiments. Mature, peptide-loaded MHC class II heterodimers remain associated in the presence of SDS detergent at room temperature (SDS-stable), whereas this treatment dissociates nonpeptide-loaded class II heterodimers. Normal SDS-stable class II heterodimers will then dissociate upon boiling. Western blot analysis using an Ab (Tu36) specific for HLA-DR{alpha}{beta} heterodimers and which does not bind free {alpha}- or {beta}-chains (38, 39, 40) demonstrates that these heterodimers are not dissociated by treatment with SDS at room temperature (Fig. 6GoA). Similarly, experiments using an HLA-DR{alpha}-specific Ab (TAL1B5) which detects monomeric HLA-DR{alpha} as well as HLA-DR-{alpha}{beta} heterodimers in Western blot (42, 45) reveal that SDS-stable heterodimers are intact in infected cells (Fig. 6GoB). However, the density of the band representing HLA-DR{alpha} free chains as well as nonpeptide-loaded heterodimers is reduced in infected cells (Fig. 6GoB). However, roughly half of eight independent replicates did not detect a decrease in the unstable and free HLA-DR{alpha} forms, suggesting the phenomenon is variable. As a negative control, a group of U373/CII cells were treated with ammonium chloride, which prevents peptide loading of MHC class II molecules (44). In all experiments, ammonium chloride-treated U373/CII cells have significantly reduced levels of SDS-stable HLA-DR heterodimers compared with noninfected and infected U373CII cells, thereby confirming the ability of the assay to detect decreases in peptide-loaded class II species. In summary, these experiments demonstrate that steady-state levels of peptide-loaded class II heterodimers are similar in noninfected and HCMV-infected U373/CII cells at 3 days after infection. However, the fraction of HLA-DR{alpha} composed of nonpeptide-loaded class II species and free {alpha}-chains is variably reduced.



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  		FIGURE 6. SDS-stable MHC class II dimers are present in HCMV-infected cells. SDS-stable Western blot analyses were performed by collecting cell lysates in Nonidet P-40 lysis buffer from noninfected and infected cells 3 days after infection, were treated with 3% SDS under nonreducing conditions for 1 h at 25°C, divided in half, either exposed to 100°C or left at room temperature, and analyzed by SDS-PAGE. Blots were probed with anti-DR{alpha}/HLA-DR{alpha}{beta} mAb Tu36, which recognizes HLA-DR{alpha}{beta} heterodimers (A); and with anti-DR{alpha} mAb TAL.1B5, which binds free HLA-DR{alpha} and HLA-DR-{alpha}/HLA-DR-{beta} heterodimers (B). Lane 3, HCMV-infected U373/CII cells contain {alpha}{beta} heterodimers resistant to treatment with SDS. Lanes 2 and 4, Boiling similarly dissociates the peptide-loaded class II heterodimers in noninfected and infected cells. Lane 5, Ammonium chloride treatment reduces levels of SDS-stable species of HLA-DR detected by both mAb Tu36 and TAL.1B5. B, Although SDS-resistant heterodimers are equivalent in HCMV-infected U373/CII cells, levels of free {alpha}-chains/nonpeptide-loaded heterodimers are reduced. (B, boiled; NB, not boiled).

 
SDS-stability experiments suggest that the block in MHC class II expression occurs downstream of class II trafficking to peptide-loading compartments. Ii molecules form a nonameric complex with class II molecules that traffic through late endosome/early lysosome compartments (noted by the presence of LAMP-1) to specific MHC class II peptide-loading compartments where the HLA-DM molecule facilitates the loading of peptides into the peptide binding groove of class II heterodimers. Confocal microscopy experiments were performed to determine whether MHC class II molecules colocalize to cellular compartments containing Ii, the LAMP-1 positive late endosome/early lysosome compartments, and finally to HLA-DM positive peptide-loading compartments in infected U373/CII cells. There was no change in the colocalization of MHC class II molecules with Ii molecules in infected U373/CII compared with noninfected controls (Fig. 7Go). Similarly, MHC class II molecules colocalize with the late endosome/early lysosome compartment marker, LAMP-1, and the peptide-loading compartment marker, HLA-DM, in infected cells (Fig. 8Go).



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  		FIGURE 7. The colocalization of MHC class II and Ii is unaltered by HCMV infection. To determine whether MHC class II and Ii are normally associated in CMV-infected U373/CII cells, we performed confocal colocalization studies. Noninfected (A, C, and E) and HCMV-infected (B, D, and F) U373/CII cells were stained 3 days after infection using dual-labeling immunofluorescence with an anti HLA-DR, DP, DQ Ab CR3/43 (green staining, A and B) and N-terminal Ii Ab Pin1.1 (red staining, C and D). The degree of colocalization between Ii and MHC class II is visualized in the merged images (yellow staining, E and F). One of three representative experiments is shown. Original magnification, x1238.

 


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  		FIGURE 8. HCMV infection does not alter the colocalization of MHC class II molecules with LAMP-1 and HLA-DM. We performed confocal microscopy studies in noninfected (A, C, E, G, I, and K) and HCMV-infected (B, D, F, H, J, and L) U373/CII cells, 3 days after infection, using dual-labeling immunofluorescence with an anti-HLA-DR, DP, DQ Ab CR3/43 (green staining, A, B, G, and H) and Abs against LAMP-1 (red staining left panels, C and D) and HLA-DM (red staining right panels, I and J). The degree of colocalization between MHC class II and LAMP-1 or HLA-DM was visualized in the merged images (yellow staining, LAMP-1/class II: E and F; HLA-DM/class II: K and L). This figure is representative of three independent experiments. Original magnification, x1238.

 
Although we found that MHC class II molecules are present in late endosomal/early lysosomal and HLA-DM positive peptide-loading compartments, the overall cellular distribution of MHC class II, Ii, LAMP-1, and HLA-DM molecules is altered in infected cells compared with noninfected controls (Figs. 7Go and 8Go). Higher magnification immunofluorescence images demonstrate that MHC class II positive vesicles are retained in a perinuclear distribution in HCMV-infected cells with a paucity of class II-associated vesicles in the cell periphery compared with noninfected controls (Fig. 9Go). This distribution predominates at 3 days after infection, the time period when the decrease in surface MHC class II expression is maximal. In these experiments, the majority of cells had qualitatively similar levels of intracellular class II as noninfected cells.



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  		FIGURE 9. HCMV infection results in the retention of MHC class II positive vesicles in a perinuclear distribution. Dual staining immunofluorescence for MHC class II molecules with CR3/43 Ab (green, A and C) and the HCMV IE protein (red, B and D) demonstrates differential patterns of class II positive vesicles in noninfected and HCMV-infected cells. In noninfected cells, MHC class II positive vesicles are distributed both in the perinuclear region and the cell periphery extending to the plasma membrane. In contrast, MHC class II positive vesicles are retained in the perinuclear region in HCMV-infected cells and there is a paucity of class II postitive vesicles in the cell periphery.

 
To localize the class of HCMV genes mediating the decrease in constitutive MHC class II surface expression, we performed experiments with an inhibitor of HCMV late gene expression, PFA. Noninfected and HCMV-infected U373/CII cells were treated with PFA and MHC class II surface expression was quantified by flow cytometry. Blocking expression of HCMV late genes does not affect the HCMV-mediated decrease in MHC class II expression, suggesting that HCMV immediate-early or early genes are responsible for this effect (Fig. 10Go).



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  		FIGURE 10. HCMV immediate early or early genes are responsible for disrupting constitutive MHC class II expression. Infected or noninfected U373/CII were treated with PFA, an inhibitor of HCMV late gene expression, and MHC class II expression was measured by flow cytometry at 0 and 3 days after infection. HCMV-infected cells not treated with PFA were used as a positive control for decreased class II. PFA treatment does not limit the HCMV-mediated decrease in MHC class II expression. *, Statistically significant decreases in class II surface levels determined by t test analysis (p < 0.001).

 
The HCMV IRS1-US11 region is a critical region of the HCMV genome containing HCMV immediate-early and early genes that have immunomodulatory functions. Specifically, this region contains the HCMV US2, US3, US6, and US11 proteins that decrease MHC class I expression (10, 11, 12, 15). Also, this region contains the US2 glycoprotein that decreases newly synthesized HLA-DR{alpha} molecule expression (33). Therefore, we tested whether the IRS1-US11 HCMV region mediates the decrease in cell surface MHC class II expression observed at 48 and 72 h after infection by performing experiments with the RV7186 HCMV strain, a deletion mutant lacking the IRS1-US11 genes (61). Flow cytometry experiments demonstrate that infection of U373/CII cells with HCMV strain RV7186 decreases MHC class II surface expression to levels approximately two-thirds that observed with wild-type CMV (Fig. 11GoA). However, as demonstrated in previous publications, the HCMV-mediated decrease in MHC class I expression is lost with RV7186 infection (Fig. 11GoA and Ref. 61). The finding that RV7186 virus is capable of decreasing MHC class II to two-thirds the levels decreased by wild-type virus, but is not capable of decreasing MHC class I expression, demonstrates that HCMV genes outside of the IRS1-US11 region mediate the majority of the decrease in cell-surface MHC class II expression. To confirm that our preparation of RV7186 was not contaminated by wild-type HCMV, PCR analyses of viral DNA demonstrates no detectable US2 in RV7186 preparations (Fig. 11GoB).



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  		FIGURE 11. The IRS1-US11 region does not mediate the decrease of cell-surface MHC class II expression in HCMV-infected U373/CII cells. A, MHC class II and MHC class I expression was analyzed by flow cytometry at 3 days after infection in noninfected U373/CII cells and U373/CII cells infected with Towne or RV7186 virus (lacking the IRS1-US11). B, PCR analysis of DNA isolated from viral stocks of Towne and RV7186 was performed for US2 and US12.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HCMV decreases cell-surface MHC class II expression in infected dendritic cells (62, 63, 64) and monocyte/macrophages, which are major targets of HCMV infection, latency, and reactivation (32, 65, 66). Interestingly, multiple mechanisms appear to mediate this decrease (64, 65, 66). To facilitate a thorough investigation of the molecular interactions of HCMV with the MHC class II Ag presentation pathway, we transfected U373 astrocytoma cells with the CIITA gene to develop a constitutive class II expression system. Herein, we demonstrate that HCMV disrupts constitutive MHC class II surface expression via a novel mechanism. HCMV IE and/or E genes, independent of the actions of a soluble factor, mediate decreased MHC class II expression on the cell surface without significantly altering steady-state class II RNA levels and only marginally decreasing steady-state HLA-DR{alpha} levels. Moreover, SDS-stability experiments reveal that peptide-loaded MHC class II heterodimers are intact in infected cells; however, the fraction of HLA-DR{alpha} composed of nonpeptide-loaded class II species and free {alpha}-chains is variably reduced.

Ii expression is not decreased in infected cells, and confocal microscopy demonstrates that Ii colocalizes with MHC class II in infected cells, arguing against a lesion in Ii-dependent class II trafficking. Further confocal analyses of the distribution of class II reveal three significant findings. First, class II molecules colocalize with LAMP-1 and HLA-DM, markers of late endosomal/early lysosomal compartments important for peptide loading. Second, there is a striking perinuclear distribution of MHC class II while class II positive vesicles are nearly absent from the cell periphery. Third, the distribution of late endocytic compartments matches that of MHC class II in HCMV-infected cells in that they reside in tight perinuclear clusters instead of being diffusely distributed throughout the cell. Therefore, in infected cells, class II molecules appear to traffic to peptide-loading compartments, but remain sequestered within these compartments in a perinuclear distribution. These results also suggest that HCMV does not alter MHC class II intracellular sorting, but rather the normal trafficking of mature class II positive vesicles toward the periphery.

Under conditions blocking HCMV late gene expression, MHC class II expression is reduced to levels seen without a restriction on viral gene expression, implicating HCMV immediate-early or early genes in this process. Experiments with the HCMV mutant strain RV7186, which lacks the IRS1-US11 genes, reveal that approximately one-third of the decrease in cell-surface MHC class II expression in infected cells at 3 days after infection is mediated by a factor contained within this viral genome segment, while the majority of the decrease in cell-surface MHC class II expression is mediated by viral genes outside of this region.

Prior studies have demonstrated that the HCMV US2 glycoprotein mediates a decrease in newly synthesized HLA-DR{alpha} and HLA-DM{alpha} expression detected by metabolic labeling and immunoprecipitation experiments (33). Herein, we detect a variable decrease in free {alpha}-chains/nonpeptide-loaded class II in HCMV-infected cells shown by steady-state SDS-stability experiments. However, steady-state HLA-DR{alpha} protein expression was only slightly decreased (~10%), and {beta} protein levels, as well as peptide-loaded HLA-DR, in our Western blot analyses were not decreased. Additionally, confocal microscopy experiments did not show a significant decrease in HLA-DR and HLA-DM expression in infected cells at 3 days after infection. Our results suggest that although HCMV US2 decreases HLA-DR{alpha} levels detected by immunoprecipitation analysis of metabolically labeled cells, it may be a relatively small decrease as compared with total cell levels of MHC class II molecules.

It is worthwhile to note that US2 expression and its relative level of class II degradation during infection may be variable. The discordant results seen in our SDS-stability experiments regarding decreases in the fraction of HLA-DR{alpha} composed of nonpeptide-loaded class II species and free {alpha}-chains may be explained by variable expression of US2. Thus, this variability as well as differences in technique may explain why Tomazin et al. (33) noted preferential destruction of HLA-DR{alpha} by US2, and we observed a predominant trafficking defect. Future experiments will be needed to resolve these issues.

The trafficking of peptide-loaded MHC class II molecules from intracellular vesicles to the surface is not well understood, but appears to involve direct trafficking of these vesicles via microtubules, with the microtubule motor kinesin mediating transport to the plasma membrane and dynein mediating retention of vesicles (67, 68). Future studies are needed to analyze the role of the actin cytoskeleton and myosin motors in regulating class II trafficking (69, 70, 71).

Our data suggest that, in addition to increasing MHC class II degradation, HCMV disrupts constitutive MHC class II surface expression by altering the intracellular distribution of MHC class II molecules and limiting their egress to the surface of infected cells. This phenomenon of altering the distribution and trafficking of class II is evident in the developmental regulation of surface MHC class II expression in dendritic cells, where during maturation, class II dramatically redistributes from intracellular lysosomal compartments to the cell surface (60, 72, 73, 74, 75). Moreover, HSV type 2 may use a similar mechanism of disrupting MHC class II trafficking as HCMV: it induces cytoplasmic and nuclear sequestering of MHC class II Ags in vivo, a phenomenon associated with lethal infection in the brains of mice (76, 77).

Cell-mediated immunity is essential in controlling HCMV infection and the CD4+ T cell contribution is a critical component (2, 3, 4, 5, 6, 7). HCMV-specific CD4+ T cells control HCMV infection through the release of IFN-{gamma} and cytolysis of infected cells in an MHC class II-restricted manner (20, 21, 22, 23). By blocking the MHC class II Ag presentation pathway in infected monocyte/macrophages or dendritic cells, HCMV may limit the exposure of virus-derived peptides to HCMV-specific CD4+ T cells, thereby aiding in the establishment of a persistent infection.

Some patient data allude to the in vivo relevance of HCMV immunosuppressive effects, and the importance of CD4+ T cells in controlling infection. HCMV infection has been shown to be an independent risk factor for fungal and bacterial superinfection in transplant recipients, often inducing high mortality rates (78, 79, 80). Moreover, in transplant patients given HCMV-specific CD8+ T cell clone therapy to control and prevent HCMV infection, cytotoxic activity declined if HCMV-specific CD4+ Th cells were deficient (81).

In conclusion, we demonstrate that HCMV uses a novel mechanism for inhibiting MHC class II expression in infected cells; mature, peptide-loaded MHC class II molecules are sequestered in endosomal/lysosomal vesicles retained in a perinuclear distribution. This effect occurs independent of a critical region of the HCMV genome that has been shown to mediate key immunomodulatory functions associated with MHC class I and class II expression. These mechanisms, combined with the HCMV-mediated blockade in IFN-stimulated responses and the MHC class I Ag presentation pathway (9, 10, 11, 12, 13, 14, 15, 17, 30), further demonstrate the remarkable diversity of HCMV immunoevasive strategies.


   Acknowledgments
 
We thank Dr. J. Boss for his gift of full-length CIITA in vector pcDNA3.1 and for helpful discussions, Dr. Thomas R. Jones for his gift of HCMV deletion mutant RV7186, Dr. P. Cresswell for his gift of Abs Pin1.1 and MaP.DM1, Dr. M. Fukuda for his gift of Ab BB6, and Joanne Trgovcich for helpful comments. We also thank Bruce Briggs for expert flow cytometry assistance; and Debbie Knight, Janny Chen, Jason Eckel, Tobie Eckel, and Ryan Carlson for their technical assistance.


   Footnotes
 
1 This research was supported by National Institutes of Health Grant RO1 AI38452-01A1. A portion of this work was presented at the 7th International Cytomegalovirus Back

2 Address correspondence and reprint requests to Dr. Daniel D. Sedmak, Department of Pathology, Ohio State University College of Medicine and Public Health, 129 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210. E-mail address: sedmak.2{at}osu.edu Back

3 Abbreviations used in this paper: HCMV, human CMV; CIITA, class II transactivator; PFA, phosphonoformic acid; Ii, invariant chain. Back

Received for publication April 2, 2001. Accepted for publication April 29, 2002.


   References
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 Materials and Methods
 Results
 Discussion
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