Constitutive MHC Class II Expression Human Cytomegalovirus Disrupts

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Human Cytomegalovirus Disrupts Constitutive MHC Class II Expression Colleen M. Cebulla, Daniel M. Miller, Yingxue Zhang, Brian M. Rahill, Peter Zimmerman, John M. Robinson and Daniel This information is current as D. Sedmak of September 24, 2021. J Immunol 2002; 169:167-176; ; doi: 10.4049/jimmunol.169.1.167 http://www.jimmunol.org/content/169/1/167 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Human Cytomegalovirus Disrupts Constitutive MHC Class II Expression1

Colleen M. Cebulla,* Daniel M. Miller,‡ Yingxue Zhang,* Brian M. Rahill,* Peter Zimmerman,* John M. Robinson,† and Daniel D. Sedmak2*

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 Downloaded from 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. The Journal of Immunology, 2002, 169: 167–176. http://www.jimmunol.org/ ytomegalovirus is an important cause of morbidity and It is well-established that CD4ϩ T cells are important in con- mortality in immunocompromised populations, e.g., trolling CMV infection. Mice depleted of CD8ϩ T cells halt dis- AIDS patients and transplant recipients (1). Human seminated CMV disease with similar kinetics as nondepleted con- C 3 ϩ CMV (HCMV), like other HSVs, is able to persist for the life of trols, suggesting a compensatory antiviral role of the CD4 the host, even in immunocompetent individuals. Reactivation of population (18). In addition, clearance of CMV from the salivary latent or persistent virus is responsible for the majority of HCMV- gland, an organ important for persistence, is dependent on CD4ϩ associated morbidity and mortality in immunosuppressed hosts. T cells (19, 20). Control of CMV replication in the salivary gland ϩ ␥ Thus, determining the factors that promote HCMV persistence is a is dependent on CD4 T cells with the TH-1 phenotype and IFN- by guest on September 24, 2021 critical step in understanding and preventing HCMV disease. is an essential factor (20). CMV-specific CD4ϩ T cells play a Cell-mediated immunity is essential in controlling HCMV and critical role in controlling CMV infection through the release of other viral infections (2–7). However, viruses have evolved re- IFN-␥, but also have been shown to mediate cytolysis of infected markable strategies for evading cell-mediated immune responses cells in an MHC class II-restricted manner (20–23). (8). Moreover, a single virus can use multiple means of blocking CD4ϩ T cells recognize viral protein-derived peptides in the Ag presentation. For example, the HCMV US2, US3, US6, and context of MHC class II molecules. MHC class II proteins are US11 gene products use distinct mechanisms to inhibit MHC class constitutively expressed in APCs such as monocyte/macrophages, I Ag presentation and escape CD8ϩ T cell responses (9–16). In dendritic cells, and B cells. MHC class II ␣- and ␤-chains form a addition, the HCMV matrix protein pp65 inhibits the presentation heterodimer in the endoplasmic reticulum and associate with the of HCMV IE1 protein-derived peptides to IE1-specific CD8ϩ T invariant chain (Ii) to form a “nonameric” complex (24–26). This cells (17). complex moves through endosomal compartments to peptide-loading compartments where the HLA-DM molecule facilitates the ex- change of the class II-associated Ii peptide portion of Ii for peptides generated in lysosomal compartments (24, 26). Class II Departments of *Pathology, and †Physiology and Cell Biology, Ohio State University molecules present these peptides to CD4ϩ T cells on the cell College of Medicine and Public Health, Columbus, OH 43210; ‡Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, FL 33136 surface. ␥ Received for publication April 2, 2001. Accepted for publication April 29, 2002. CMV blocks IFN- -stimulated MHC class II expression in endothelial cells, an important site of CMV infection in vivo (27, 28). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance HCMV inhibits IFN-␥ inducible MHC class II expression by with 18 U.S.C. Section 1734 solely to indicate this fact. blocking the JAK/STAT signal transduction pathway in infected 1 This research was supported by National Institutes of Health Grant RO1 AI38452- cells and murine CMV blocks IFN-␥-stimulated MHC class II ex- 01A1. A portion of this work was presented at the 7th International Cytomegalovirus Workshop. C.M.C. was a Presidential Fellow at Ohio State University. D.M.M. was pression through a JAK/STAT-independent mechanism (29–31). a Howard Hughes Medical Institute Predoctoral Fellow. HCMV infection decreases constitutive MHC class II expres- 2 Address correspondence and reprint requests to Dr. Daniel D. Sedmak, Department sion in monocyte/macrophages, a critical site of HCMV latency of Pathology, Ohio State University College of Medicine and Public Health, 129 and reactivation (32). There is evidence that the HCMV US2 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210. E-mail address: [email protected] glycoprotein, a molecule previously shown to target MHC class ␣ 3 Abbreviations used in this paper: HCMV, human CMV; CIITA, class II transacti- I H chains for degradation, mediates a decrease in HLA-DR vator; PFA, phosphonoformic acid; Ii, invariant chain. and HLA-DM expression at early times after infection (33).

Given the fact that HCMV uses four distinct gene products to GATCCAGTTTGCAC-3Ј) and antisense (5Ј-GCCCTAGGATATTTAT block MHC class I expression and that these gene products GAAAAAGCCAGTGTGCC-3Ј). target distinct levels of the class I Ag processing pathway, we Northern blot analysis suspected additional, US2-independent mechanisms for the decrease in MHC class II expression in infected cells. 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 chlo- Herein, we investigate the HCMV-mediated disruption of con- ride centrifugation method (36). The RNA was separated on a 1.4% aga- stitutive MHC class II expression by generating a model system rose 0.22-M formaldehyde gel. Northern blots were probed for HLA-DR␣ that facilitates molecular analyses. Monocyte/macrophages, which as well as the loading control GAPDH. Probes were generated using ran- 32 are an important site of HCMV infection in vivo and constitutively dom priming and [␣- P]dCTP (Deca Prime kit; Ambion, Austin, TX). ␣ express MHC class II, are not efficiently infected in vitro, thereby cDNA templates were generated for the HLA-DR probes using primer sets sense (5Ј-AAAGCGCTCCAACTATACTCCGA-3Ј) and antisense: limiting investigations of the molecular mechanism for decreased (5Ј-ACCCTGCAGTCGTAAACGTCC-3Ј) and clone p0A1 (ATCC) was class II in these primary cells. To generate a model to study the used for the GAPDH probes. Dilutional standards of 10, 5, and 2.5 ␮g interaction of HCMV with MHC class II, we transfected a class II RNA isolated from noninfected and HCMV-infected U373/CII were run as negative, HCMV permissive cell line, U373 astrocytoma cells, controls. Densitometry analyses were performed by scanning autoradiographic films with a Hewlett Packard flatbed scanner (Hewlett Packard, with class II transactivator (CIITA) cDNA, thereby creating cells Palo Alto, CA), and the digital images were analyzed using Scion Image that constitutively express class II molecules. CIITA is the “master software (Scion Corporation, Frederick, MD). switch” in class II expression, and transfection of CIITA into class II negative cells results in constitutive MHC class II, Ii, and Flow cytometry HLA-DM expression (34). U373 cells are permissive for HCMV Cells were harvested with trypsin/EDTA, labeled with directly conjugated Downloaded from infection and have been used for investigating HCMV/MHC class fluorescein-labeled Abs, and washed three times with SBSS. Abs for I interactions (10–13, 33). Using this model system, we have dis- monomorphic HLA-DR epitopes (clone Tu36 from GenTrak (Plymouth Meeting, PA; Refs. 38–40); and L243 (41) from BD PharMingen, San covered a mechanism for the HCMV-mediated disruption of MHC Diego, CA) were used as well as MHC class I (anti-HLA-A, B, C; ICN class II expression that is independent of the effects of US2 on Biomedicals, Irvine, CA). A minimum of 5 ϫ 103 cells were analyzed on MHC class II proteins. a Coulter flow cytometer (Coulter, Hialeah, FL) calibrated with 2% fluo-

rescent microbeads (Standard Brite Beads; Coulter). Nonspecific fluores- http://www.jimmunol.org/ cence was assessed by staining cells with an isotype control Ab. Mean Materials and Methods fluorescence intensity derived from a linear scale was reported by subtract- Cells and transfections ing nonspecific mean fluorescence intensity values from MHC class I or class II mean fluorescence intensity values. U373/CII cells were generated by stable transfection of U373 cells (Amer- ican Type Culture Collection, Manassas, VA) with full-length CIITA Immunoprecipitation, Western analysis, and SDS-stability assay cDNA (a generous gift from Dr. J. Boss, Emory University School of Medicine, Atlanta, GA), driven by the CMV IE promoter, in vector pcDNA For HLA-DR␣ and Ii immunoprecipitations, U373/CII and U373/pc cells 3.1 using SuperFect reagent (Qiagen, Valencia, CA). Stable transfectants were treated with lysis buffer containing 1% Triton X-100, 0.15 M NaCl, were selected by adding 400 ␮g/ml of G418 (Life Technologies, Grand 50 mM Tris (pH 8.0), 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM ␮ Island, NY) to culture media (DMEM with 10% FBS). FACS sorting was PMSF, 1 mM orthovanadate, and 5 g/ml each of pepstatin, leupeptin, and by guest on September 24, 2021 performed on stably transfected cells and only cells with high levels of aprotinin. Samples were sonicated and precleared with an isotypic control HLA-DR expression were collected. An individual clone, U373/CII, with Ab and protein G-Sepharose. The cleared supernatants were incubated with high HLA-DR surface levels was isolated with cloning rings. Control cells, primary Ab, TAL.1B5 (HLA-DR␣; DAKO, Glostrup, Denmark) (42), LN2 U373/pc, were generated by transfecting U373 cells with the pcDNA 3.1 (Ii C terminus; BD PharMingen) or Pin1.1 (Ii N terminus; a generous gift vector with no insert and selecting for stable transfection with G418. of Dr. P. Cresswell, Yale University School of Medicine, New Haven, CT) (43). Immune complexes were collected with protein G-Sepharose, pel- CMV infection and viral stock preparation leted by centrifugation, washed in lysis buffer, and resuspended in SDS- PAGE loading buffer containing 0.2% 2-ME (36). Samples were boiled 5 U373/CII were infected with Towne HCMV, AD169 HCMV, or the min, Sepharose beads pelleted, and precipitated proteins fractionated by HCMV deletion mutant RV7186, which is deleted for IRS1-US11 (35) at 12% SDS-PAGE. Western blot analyses used the same lysis buffer as for a multiplicity of infection of 10. Infection levels were confirmed by HCMV immunoprecipitation experiments. Samples were diluted 1/1 in sample IE immunofluorescence (clone MAB810; Chemicon International, Te- buffer, boiled, and loaded on 12% SDS-PAGE gels. mecula, CA) and were 80–90%. For analysis of HCMV gene expression, For SDS-stability experiments, a group of cells were treated with 50 U373/CII were infected in the presence of 300 ␮g/ml phosphonoformic mM NH4Cl, to inhibit peptide loading of MHC class II as a negative con- acid (PFA), an inhibitor of HCMV late gene expression. trol (44). SDS-stability lysis buffer containing 0.5% Nonidet P-40, 0.15 M Cell-free viral stocks of HCMV were prepared as previously described NaCl, 50 mM Tris (pH 8.0), and protease inhibitors was used. Samples (30). Briefly, virus was propagated in fibroblasts (MRC-5), passages 22– were treated with 3% SDS for1hat25°C under nonreducing conditions, 35, at low multiplicity of infection. Virions were stored frozen at Ϫ80°C then split in half and either heated to 100°C or maintained at room tem- and standard plaque assays were used to quantitate viral titers from rep- perature for 5 min. Samples were then loaded on 12% SDS-PAGE gels, resentative vials. transferred to nitrocellulose, and blocked in 5% nonfat dry milk. Immunoblotting was performed using Abs: TAL.1B5 (HLA-DR␣, DAKO; RT-PCR Refs. 42 and 45), DK22 (code M704, recognizing ␤-chain of HLA-DR, DP, and DQ; DAKO) (46, 47), TU36 (recognizing HLA-DR␣␤ complexes; Caltag Total RNA isolated using guanidine thiocyanate extraction and cesium Laboratories, Burlingame, CA) (38–40), LN2 (Ii C terminus, BD PharMin- chloride centrifugation (36) was reverse transcribed into cDNA using the gen), or Pin1.1 (Ii N terminus, a generous gift of Dr. P. Cresswell; Ref. 43), SuperScript Preamplification System (Life Technologies), and 500 ng followed by anti-mouse IgG-HRP secondary Ab, or polyclonal rabbit anti- cDNA was used per PCR. PCR (50 ␮l) were performed using PCR buffer STAT-2 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-rabbit IgG- (20 mM Tris-HCL (pH 8.4), 50 mM KCl), 1.5 mM MgCl , 20 mM dNTP, 2 HRP secondary Ab (Santa Cruz Biotechnology). Blots were developed using 400 nM of each sense and antisense primer, and 5 U Taq polymerase (Life ECL (Amersham Pharmacia Biotech, Piscataway, NJ) or West Dura Super Technologies). PCR were incubated for 20 cycles on an Ericomp Easy Signal (Pierce, Rockford, IL) chemiluminescence detection systems. Densi- Cycler (Ericomp, San Diego, CA). Previously published CIITA primers tometry analyses of Western and immunoprecipitation films were performed were used (37). The ␤-actin primers used were sense (5Ј-GTGGGGCGC as for Northern blot autoradiographic films. CCCAGGCACCA-3Ј) and antisense (5Ј-CTCCTTAATGTCACGCAC GATTTC-3Ј). PCR analysis of US2 and US12 was performed on phenol: Immunofluorescence and confocal microscopy chloroform-extracted DNA (36) isolated from viral stocks of Towne and RV7186. The primer sets used for US2 were sense (5Ј-ATGAACAA Infected and noninfected U373/CII cells were grown on glass chamber- TCTCTGGAAAGCC-3Ј) and antisense (5Ј-TCAGCACACGAAAAAC slides (Nunc, Naperville, IL), then fixed using a modification of Muczynski CGCAT-3Ј), and for US12 were sense (5Ј-CGGAATTCATGGTACA et al. (48). Briefly, at 3 days after infection, cells were rinsed with PBS/30 The Journal of Immunology 169 mM sucrose, fixed for 10 min with 4% paraformaldehyde, and permeabil- Results ized with 0.2% saponin (in PBS/30 mM sucrose/1% BSA). Cells were Cells that constitutively express MHC class II molecules (U373/ stained as follows: anti-CMV IE Ab (clone MAB810, Chemicon Interna- tional), lysosmal-associated membrane protein 1 for late endosomes/early CII) were generated by transfecting class II negative U373 cells lysosomes (clone BB6, a generous gift of Dr. M. Fukuda, La Jolla Cancer with CIITA cDNA, FACS sorting for high MHC class II expres- Research Center, Burnham Institute, La Jolla, CA; Ref. 49), HLA-DM sion, and isolating an individual clone with cloning rings. Fig. 1A (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 shows HLA-DR expression in the original CIITA-transfected pop- Ј were followed by a goat-anti-mouse F(ab )2 secondary Ab conjugated to ulation of U373 cells and high levels of HLA-DR expression in the Rhodamine Red-X (Jackson ImmunoResearch Laboratories, West Grove, isolated U373/CII clone (Fig. 1A). Stable transfection was con- PA). For dual labeling, cells were stained with an Ab recognizing HLA- firmed by RT-PCR analysis (Fig. 1B). 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- To analyze the effects of CMV infection on surface MHC class CMV IE, and secondary Ab alone was used as a negative control for II expression, U373/CII were infected with the Towne strain of LAMP-1, HLA-DM, and Pin1.1. Results were analyzed on a Bio-Rad HCMV and surface HLA-DR expression was measured by flow MRC 600 confocal microscope equipped with an argon-krypton laser (Bio- cytometry. HCMV infection decreased HLA-DR levels by 2 days Rad, Hercules, CA). Excitation wavelengths were 488 and 568 nm, and ϭ ϭ detection wavelengths were 522 and 585 for FITC and rhodamine visual- after infection ( p 0.01), with a maximal decrease at 3 days ( p ization, respectively. For dual localization, the images were merged using 0.004), yielding a 60% reduction (Ϯ5%) in class II molecules on Confocal Assistant software (public domain software developed by T. C. the surface of infected cells (Fig. 2A). Similar results were ob- Brelje, University of Minnesota, Minneapolis, MN). tained using two anti-HLA-DR mAbs reactive to distinct portions

Statistics of the HLA-DR molecule as well as infection with the AD169 Downloaded from CMV strain (data not shown). As appropriate, data are shown as the mean Ϯ SEM. One-sided Student’s t tests were used to determine signiﬁcant differences between groups (Sig- The CMV US2, US3, US6, and US11 glycoproteins mediate a maStat Statistical Analysis system; SPSS, Chicago, IL). decrease in MHC class I expression (9–16). To conﬁrm that a http://www.jimmunol.org/ by guest on September 24, 2021

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 ϳ75% 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. 170 HCMV DISRUPTS CONSTITUTIVE MHC CLASS II

FIGURE 3. HCMV infection does not decrease HLA-DR␣ mRNA levels in U373/CII. A, Northern blot analysis of HLA-DR␣ RNA levels was

performed with U373/CII at 3, 24, 48, and 72 h after infection. GAPDH Downloaded from was probed as a loading control. HCMV infection does not alter HLA-DR␣ RNA levels at 3 days after infection. B, Dilutional Northern analysis of noninfected and infected U373/CII HLA-DR␣ RNA levels at 3 days after infection was performed to conﬁrm the detection range of the assay.

Analysis of HLA-DR␣ and HLA-DR␤ protein expression by http://www.jimmunol.org/ FIGURE 2. HCMV infection decreases constitutive MHC class II sur- Western blot analysis at 3 days after infection demonstrates no face expression. U373/CII cells were infected with HCMV and cell surface ␤ MHC class II expression was analyzed by flow cytometry. A, HCMV in- change in steady-state levels of HLA-DR and only a slight de- ␣ fection results in decreased HLA-DR expression beginning 2 days after crease in HLA-DR molecules (8%) by scanning densitometry compared with the levels of an internal control, STAT-2 with ,ء .infection with the lowest levels detected at three days after infection Statistically significant differences compared with the day 0 control (p ϭ HCMV infection (Fig. 4). Furthermore, detection of steady-state 0.01 for 2 days after infection, and p ϭ 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 by guest on September 24, 2021 I) expression. 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. 2B). 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␣ mRNA at 3 FIGURE 4. Total cellular levels of MHC class II proteins are not sig- days after HCMV infection, despite the significant decrease in sur- nificantly decreased by HCMV infection. A, Western blot analysis of total cell levels of HLA-DR␣ and HLA-DR␤ at 3 days after infection in U373/ face HLA-DR expression at this time, suggesting that the decrease CII was performed. There is no significant decrease in the steady-state in class II expression is not due to alterations in steady-state MHC levels of HLA-DR␤ protein in HCMV-infected cells at 3 days after infec- class II RNA (Fig. 3A). Dilutional Northern blot analyses of tion compared with the levels of an internal control STAT-2. B, There is HCMV-infected and noninfected U373/CII 3 days after infection only a slight decrease in steady-state HLA-DR␣ levels (8% as determined confirmed that differences in HLA-DR␣ mRNA levels in infected by denistomtery). Ab TAL.1B5 was used to detect HLA-DR␣ and DK22 and noninfected cells were detectable with this assay (Fig. 3B). used to detect HLA-DR␤. The Journal of Immunology 171

HLA-DR␣ by immunoprecipitation followed by immunoblotting yields identical results. 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 (54–60). 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. 5) or N-terminal-specific Ab (data not shown). Although there is not a quantitative decrease of Ii in HCMV- infected cells, qualitative defects in Ii function may alter the qual- ity 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 de- FIGURE 6. SDS-stable MHC class II dimers are present in HCMV- Downloaded from infected cells. SDS-stable Western blot analyses were performed by col- termine the maturation state of class II molecules in infected cells, lecting cell lysates in Nonidet P-40 lysis buffer from noninfected and in- we performed SDS-stability Western blot experiments. Mature, fected cells 3 days after infection, were treated with 3% SDS under peptide-loaded MHC class II heterodimers remain associated in nonreducing conditions for1hat25°C, divided in half, either exposed to the presence of SDS detergent at room temperature (SDS-stable), 100°C or left at room temperature, and analyzed by SDS-PAGE. Blots whereas this treatment dissociates nonpeptide-loaded class II het- were probed with anti-DR␣/HLA-DR␣␤ mAb Tu36, which recognizes erodimers. Normal SDS-stable class II heterodimers will then dis- HLA-DR␣␤ heterodimers (A); and with anti-DR␣ mAb TAL.1B5, which http://www.jimmunol.org/ sociate upon boiling. Western blot analysis using an Ab (Tu36) binds free HLA-DR␣ and HLA-DR-␣/HLA-DR-␤ heterodimers (B). Lane specific for HLA-DR␣␤ heterodimers and which does not bind 3, HCMV-infected U373/CII cells contain ␣␤ heterodimers resistant to free ␣-or␤-chains (38–40) demonstrates that these heterodimers treatment with SDS. Lanes 2 and 4, Boiling similarly dissociates the pep- are not dissociated by treatment with SDS at room temperature tide-loaded class II heterodimers in noninfected and infected cells. Lane 5, Ammonium chloride treatment reduces levels of SDS-stable species of (Fig. 6A). Similarly, experiments using an HLA-DR␣-specificAb ␣ HLA-DR detected by both mAb Tu36 and TAL.1B5. B, Although SDS- (TAL1B5) which detects monomeric HLA-DR as well as HLA- resistant heterodimers are equivalent in HCMV-infected U373/CII cells, ␣␤ DR- heterodimers in Western blot (42, 45) reveal that SDS- levels of free ␣-chains/nonpeptide-loaded heterodimers are reduced. (B, stable heterodimers are intact in infected cells (Fig. 6B). However, boiled; NB, not boiled). the density of the band representing HLA-DR␣ free chains as well by guest on September 24, 2021 as nonpeptide-loaded heterodimers is reduced in infected cells (Fig. 6B). However, roughly half of eight independent replicates did not detect a decrease in the unstable and free HLA-DR␣ forms, group of U373/CII cells were treated with ammonium chloride, suggesting the phenomenon is variable. As a negative control, a 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␣ composed of nonpeptide-loaded class II species and free ␣-chains is variably reduced. 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 FIGURE 5. Total cellular levels of Ii are not decreased by HCMV in- LAMP-1 positive late endosome/early lysosome compartments, fection. Immunoprecipitation of Ii chain reveals that HCMV infection does not decrease the relative levels of the three Ii isoforms: p41, p35, and p33. and finally to HLA-DM positive peptide-loading compartments in This result was observed with Abs to the C terminus of Ii. Comparable infected U373/CII cells. There was no change in the colocalization results were obtained with an N terminus-binding Ii Ab. Equivalent pro- of MHC class II molecules with Ii molecules in infected U373/CII portion of lysate for the three lanes was verified by analyzing STAT-2 compared with noninfected controls (Fig. 7). Similarly, MHC class expression as an internal control (data not shown). II molecules colocalize with the late endosome/early lysosome 172 HCMV DISRUPTS CONSTITUTIVE MHC CLASS II

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 experi- Downloaded from ments is shown. Original magniﬁcation, ϫ1238. http://www.jimmunol.org/ compartment marker, LAMP-1, and the peptide-loading compart- time period when the decrease in surface MHC class II expression ment marker, HLA-DM, in infected cells (Fig. 8). is maximal. In these experiments, the majority of cells had qual- Although we found that MHC class II molecules are present in itatively similar levels of intracellular class II as noninfected cells. late endosomal/early lysosomal and HLA-DM positive peptide- To localize the class of HCMV genes mediating the decrease in loading compartments, the overall cellular distribution of MHC constitutive MHC class II surface expression, we performed ex- class II, Ii, LAMP-1, and HLA-DM molecules is altered in infected periments with an inhibitor of HCMV late gene expression, PFA.

cells compared with noninfected controls (Figs. 7 and 8). Higher Noninfected and HCMV-infected U373/CII cells were treated with by guest on September 24, 2021 magnification immunofluorescence images demonstrate that MHC PFA and MHC class II surface expression was quantified by flow class II positive vesicles are retained in a perinuclear distribution cytometry. Blocking expression of HCMV late genes does not af- in HCMV-infected cells with a paucity of class II-associated ves- fect the HCMV-mediated decrease in MHC class II expression, icles in the cell periphery compared with noninfected controls (Fig. suggesting that HCMV immediate-early or early genes are respon- 9). This distribution predominates at 3 days after infection, the sible for this effect (Fig. 10).

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, ϫ1238. The Journal of Immunology 173 Downloaded from

FIGURE 9. HCMV infection results in the retention of MHC class II positive vesicles in a perinuclear distribution. Dual staining immunoﬂuo- rescence 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 http://www.jimmunol.org/ 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.

The HCMV IRS1-US11 region is a critical region of the HCMV genome containing HCMV immediate-early and early genes that by guest on September 24, 2021 have immunomodulatory functions. Speciﬁcally, this region con- FIGURE 11. The IRS1-US11 region does not mediate the decrease of tains the HCMV US2, US3, US6, and US11 proteins that decrease cell-surface MHC class II expression in HCMV-infected U373/CII cells. A, MHC class I expression (10–12, 15). Also, this region contains the MHC class II and MHC class I expression was analyzed by ﬂow cytometry US2 glycoprotein that decreases newly synthesized HLA-DR␣ 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.

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. 11A). However, as demonstrated in previous publications, the HCMV-mediated decrease in MHC class I expression is lost with RV7186 infection (Fig. 11A and Ref. 61). The finding that RV7186 virus is capable of FIGURE 10. HCMV immediate early or early genes are responsible for decreasing MHC class II to two-thirds the levels decreased by disrupting constitutive MHC class II expression. Infected or noninfected wild-type virus, but is not capable of decreasing MHC class I U373/CII were treated with PFA, an inhibitor of HCMV late gene expres- expression, demonstrates that HCMV genes outside of the sion, and MHC class II expression was measured by flow cytometry at 0 IRS1-US11 region mediate the majority of the decrease in cell- 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 surface MHC class II expression. To confirm that our prepara- ,Statis- tion of RV7186 was not contaminated by wild-type HCMV ,ء .limit the HCMV-mediated decrease in MHC class II expression tically significant decreases in class II surface levels determined by t test PCR analyses of viral DNA demonstrates no detectable US2 in analysis (p Ͻ 0.001). RV7186 preparations (Fig. 11B). 174 HCMV DISRUPTS CONSTITUTIVE MHC CLASS II

Discussion It is worthwhile to note that US2 expression and its relative level HCMV decreases cell-surface MHC class II expression in infected of class II degradation during infection may be variable. The dis- dendritic cells (62–64) and monocyte/macrophages, which are ma- cordant results seen in our SDS-stability experiments regarding jor targets of HCMV infection, latency, and reactivation (32, 65, decreases in the fraction of HLA-DR␣ composed of nonpeptide- ␣ 66). Interestingly, multiple mechanisms appear to mediate this de- loaded class II species and free -chains may be explained by crease (64–66). To facilitate a thorough investigation of the mo- variable expression of US2. Thus, this variability as well as dif- lecular interactions of HCMV with the MHC class II Ag presen- ferences in technique may explain why Tomazin et al. (33) noted ␣ tation pathway, we transfected U373 astrocytoma cells with the preferential destruction of HLA-DR by US2, and we observed a CIITA gene to develop a constitutive class II expression system. predominant trafficking defect. Future experiments will be needed Herein, we demonstrate that HCMV disrupts constitutive MHC to resolve these issues. class II surface expression via a novel mechanism. HCMV IE The trafficking of peptide-loaded MHC class II molecules from and/or E genes, independent of the actions of a soluble factor, intracellular vesicles to the surface is not well understood, but mediate decreased MHC class II expression on the cell surface appears to involve direct trafficking of these vesicles via mi- without significantly altering steady-state class II RNA levels and crotubules, with the microtubule motor kinesin mediating trans- only marginally decreasing steady-state HLA-DR␣ levels. More- port to the plasma membrane and dynein mediating retention of over, SDS-stability experiments reveal that peptide-loaded MHC vesicles (67, 68). Future studies are needed to analyze the role class II heterodimers are intact in infected cells; however, the frac- of the actin cytoskeleton and myosin motors in regulating class II trafficking (69–71). tion of HLA-DR␣ composed of nonpeptide-loaded class II species Our data suggest that, in addition to increasing MHC class II Downloaded from and free ␣-chains is variably reduced. degradation, HCMV disrupts constitutive MHC class II surface Ii expression is not decreased in infected cells, and confocal expression by altering the intracellular distribution of MHC class microscopy demonstrates that Ii colocalizes with MHC class II in II molecules and limiting their egress to the surface of infected infected cells, arguing against a lesion in Ii-dependent class II traf- cells. This phenomenon of altering the distribution and trafficking ficking. Further confocal analyses of the distribution of class II of class II is evident in the developmental regulation of surface reveal three significant findings. First, class II molecules colocalize MHC class II expression in dendritic cells, where during matura- http://www.jimmunol.org/ with LAMP-1 and HLA-DM, markers of late endosomal/early ly- tion, class II dramatically redistributes from intracellular lysoso- sosomal compartments important for peptide loading. Second, mal compartments to the cell surface (60, 72–75). Moreover, HSV there is a striking perinuclear distribution of MHC class II while type 2 may use a similar mechanism of disrupting MHC class II class II positive vesicles are nearly absent from the cell periphery. trafficking as HCMV: it induces cytoplasmic and nuclear seques- Third, the distribution of late endocytic compartments matches that tering of MHC class II Ags in vivo, a phenomenon associated with of MHC class II in HCMV-infected cells in that they reside in tight lethal infection in the brains of mice (76, 77). perinuclear clusters instead of being diffusely distributed through- Cell-mediated immunity is essential in controlling HCMV in- out the cell. Therefore, in infected cells, class II molecules appear fection and the CD4ϩ T cell contribution is a critical component to traffic to peptide-loading compartments, but remain sequestered (2–7). HCMV-specific CD4ϩ T cells control HCMV infection by guest on September 24, 2021 within these compartments in a perinuclear distribution. These re- through the release of IFN-␥ and cytolysis of infected cells in an sults also suggest that HCMV does not alter MHC class II intra- MHC class II-restricted manner (20–23). By blocking the MHC cellular sorting, but rather the normal trafficking of mature class II class II Ag presentation pathway in infected monocyte/macro- positive vesicles toward the periphery. phages or dendritic cells, HCMV may limit the exposure of virus- Under conditions blocking HCMV late gene expression, MHC derived peptides to HCMV-specific CD4ϩ T cells, thereby aiding class II expression is reduced to levels seen without a restriction on in the establishment of a persistent infection. viral gene expression, implicating HCMV immediate-early or Some patient data allude to the in vivo relevance of HCMV early genes in this process. Experiments with the HCMV mutant immunosuppressive effects, and the importance of CD4ϩ T cells in strain RV7186, which lacks the IRS1-US11 genes, reveal that ap- controlling infection. HCMV infection has been shown to be an proximately one-third of the decrease in cell-surface MHC class II independent risk factor for fungal and bacterial superinfection in expression in infected cells at 3 days after infection is mediated by transplant recipients, often inducing high mortality rates (78– a factor contained within this viral genome segment, while the 80). Moreover, in transplant patients given HCMV-specific majority of the decrease in cell-surface MHC class II expression is CD8ϩ T cell clone therapy to control and prevent HCMV in- mediated by viral genes outside of this region. fection, cytotoxic activity declined if HCMV-specific CD4ϩ Th Prior studies have demonstrated that the HCMV US2 glycop- cells were deficient (81). rotein mediates a decrease in newly synthesized HLA-DR␣ and In conclusion, we demonstrate that HCMV uses a novel mech- HLA-DM␣ expression detected by metabolic labeling and immu- anism for inhibiting MHC class II expression in infected cells; noprecipitation experiments (33). Herein, we detect a variable de- mature, peptide-loaded MHC class II molecules are sequestered in crease in free ␣-chains/nonpeptide-loaded class II in HCMV-in- endosomal/lysosomal vesicles retained in a perinuclear distribu- fected cells shown by steady-state SDS-stability experiments. tion. This effect occurs independent of a critical region of the However, steady-state HLA-DR␣ protein expression was only HCMV genome that has been shown to mediate key immuno- slightly decreased (ϳ10%), and ␤ protein levels, as well as pep- modulatory functions associated with MHC class I and class II tide-loaded HLA-DR, in our Western blot analyses were not de- expression. These mechanisms, combined with the HCMV-medi- creased. Additionally, confocal microscopy experiments did not ated blockade in IFN-stimulated responses and the MHC class I show a significant decrease in HLA-DR and HLA-DM expression Ag presentation pathway (9–15, 17, 30), further demonstrate the in infected cells at 3 days after infection. Our results suggest that remarkable diversity of HCMV immunoevasive strategies. although HCMV US2 decreases HLA-DR␣ levels detected by immunoprecipitation analysis of metabolically labeled cells, it may Acknowledgments be a relatively small decrease as compared with total cell levels of We thank Dr. J. Boss for his gift of full-length CIITA in vector pcDNA3.1 MHC class II molecules. and for helpful discussions, Dr. Thomas R. Jones for his gift of HCMV The Journal of Immunology 175 deletion mutant RV7186, Dr. P. Cresswell for his gift of Abs Pin1.1 and 28. Sedmak, D. D., S. Chaiwiriyakul, D. A. Knight, and W. J. Waldman. 1995. The ␤ MaP.DM1, Dr. M. Fukuda for his gift of Ab BB6, and Joanne Trgovcich role of interferon in cytomegalovirus-mediated inhibition of HLA-DR induction. Arch. Virol. 140:111. for helpful comments. We also thank Bruce Briggs for expert flow cytom- 29. Ng-Bautista, C. L., and D. D. Sedmak. 1995. Cytomegalovirus infection is as- etry assistance; and Debbie Knight, Janny Chen, Jason Eckel, Tobie Eckel, sociated with absence of alveolar epithelial cell HLA class II antigen expression. and Ryan Carlson for their technical assistance. J. Infect. Dis. 171:39. 30. Miller, D. M., B. M. Rahill, J. M. Boss, M. D. Lairmore, J. E. Durbin, References W. J. Waldman, and D. D. Sedmak. 1998. Human cytomegalovirus inhibits major histocompatibility complex class II expression by disruption of the Jak/Stat path- 1. Britt, W. J. 1994. Infections associated with human cytomegalovirus. In Herpes- way. J. Exp. Med. 187:675. virus Infections. R. Glaser and J. E. Jones, eds. Marcel Dekker, New York, p. 59. 31. Heise, M. T., M. Connick, and H. W. Virgin. 1998. Murine cytomegalovirus 2. Reddehase, M. J., W. Mutter, K. Munch, H. J. Buhring, and U. H. Koszinowski. inhibits interferon ␥-induced antigen presentation to CD4 T cells by macrophages 1987. CD8-positive T lymphocytes specific for murine cytomegalovirus imme- via regulation of expression of major histocompatibility complex class II-asso- diate-early antigens mediate protective immunity. J. Virol. 61:3102. ciated genes. J. Exp. Med. 187:1037. 3. Bukowski, J. F., B. A. Woda, S. Habu, K. Okumura, and R. M. Welsh. 1983. 32. Fish, K. N., W. Britt, and J. A. Nelson. 1996. A novel mechanism for persistence Natural killer cell depletion enhances virus synthesis and virus-induced hepatitis of human cytomegalovirus in macrophages. J. Virol. 70:1855. in vivo. J. Immunol. 131:1531. 33. Tomazin, R., J. Boname, N. R. Hegde, D. M. Lewinsohn, Y. Altschuler, 4. Tay, C. H., and R. M. Welsh. 1997. Distinct organ-dependent mechanisms for the T. R. Jones, P. Cresswell, J. A. Nelson, S. R. Riddell, and D. C. Johnson. 1999. control of murine cytomegalovirus infection by natural killer cells. J. Virol. 71: Cytomegalovirus US2 destroys two components of the MHC class II pathway, 267. preventing recognition by CD4ϩ T cells. Nat. Med. 5:1039. 5. Biron, C. A. 1994. Cytokines in the generation of immune responses to, and 34. Boss, J. M. 1997. Regulation of transcription of MHC class II genes. Curr. Opin. resolution of, virus infection. Curr. Opin. Immunol. 6:530. Immunol. 9:107. 6. Orange, J. S., and C. A. Biron. 1996. Characterization of early IL-12, IFN-␣␤, 35. Jones, T. R., and V. P. Muzithras. 1992. A cluster of dispensable genes within the and TNF effects on antiviral state and NK cell responses during murine cyto- human cytomegalovirus genome short component: IRS1, US1 through US5, and megalovirus infection. J. Immunol. 156:4746.

the US6 family. J. Virol. 66:2541. Downloaded from 7. Biron, C. A. 1997. Activation and function of natural killer cell responses during 36. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Sidman, J. A. Smith, viral infections. Curr. Opin. Immunol. 9:24. and K. Struhl. 1991. Current Protocols in Molecular Biology. Wiley, New York. 8. Miller, D. M., and D. D. Sedmak. 1999. Viral effects on antigen processing. Curr. 37. Brown, J. A., X. F. He, S. D. Westerheide, and J. M. Boss. 1995. Characterization Opin. Immunol. 11:94. of the expressed CIITA allele in the class II MHC transcriptional mutant RJ2.2.5. 9. Ahn, K., A. Angulo, P. Ghazal, P. A. Peterson, Y. Yang, and K. Fruh. 1996. Immunogenetics 43:88. Human cytomegalovirus inhibits antigen presentation by a sequential multistep 38. Ziegler, A., J. Heinig, C. Muller, H. Gotz, F. P. Thinnes, B. Uchanska-Ziegler, process. Proc. Natl. Acad. Sci. USA 93:10990. and P. Wernet. 1986. Analysis by sequential immunoprecipitations of the spec- 10. Jones, T. R., E. J. H. J. Wiertz, L. Sun, K. N. Fish, J. A. Nelson, and H. L. Ploegh. iﬁcities of the monoclonal antibodies TU22, 34, 35, 36, 37, 39, 43, 58 and YD1/

1996. Human cytomegalovirus US3 impairs transport and maturation of major http://www.jimmunol.org/ 63.HLK directed against human HLA class II antigens. Immunobiology 171:77. histocompatibility complex class I heavy chains. Proc. Natl. Acad. Sci. USA 39. Bijlmakers, M. J., P. Benaroch, and H. L. Ploegh. 1994. Assembly of HLA DR1 93:11327. molecules translated in vitro: binding of peptide in the endoplasmic reticulum 11. Wiertz, E. J. H. J., D. Tortorella, M. Bogyo, J. Yu, W. Mothes, T. R. Jones, precludes association with invariant chain. EMBO J. 13:2699. T. A. Rapoport, and H. L. Ploegh. 1996. Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction. Nature 40. Stumptner-Cuvelette, P., S. Morchoisne, M. Dugast, S. Le Gall, G. Raposo, 384:432. O. Schwartz, and P. Benaroch. 2001. HIV-1 nef impairs MHC class II antigen 12. Wiertz, E. J. H. J., T. R. Jones, L. Sun, M. Bogyo, H. J. Gueze, and H. L. Ploegh. presentation and surface expression. Proc. Natl. Acad. Sci. USA 98:12144. 1996. The human cytomegalovirus US11 gene product dislocates MHC class I 41. Lampson, L. A., and R. Levy. 1980. Two populations of Ia-like molecules on a heavy chains from the endoplasmic reticulum. Cell 84:769. human cell line. J. Immunol. 125:293. 13. Machold, R. P., E. J. Wiertz, T. R. Jones, and H. L. Ploegh. 1997. The HCMV 42. Adams, T. E., J. G. Bodmer, and W. F. Bodmer. 1983. Production and charac- ␣ gene products US11 and US2 differ in their ability to attack allelic forms of terization of monoclonal antibodies recognizing the -chain subunits of human Ia alloantigens. Immunology 50:613. murine major histocompatibility complex (MHC) class I heavy chains. J. Exp. by guest on September 24, 2021 Med. 185:363. 43. Lamb, C. A., and P. J. Cresswell. 1992. Assembly and transport properties of 14. Schust, D. J., D. Tortorella, J. Seebach, C. Phan, and H. L. Ploegh. 1998. Tro- invariant chain trimers and HLA-DR-invariant chain complexes. J. Immunol. phoblast class I major histocompatibility complex (MHC) products are resistant 148:3478. to rapid degradation imposed by the human cytomegalovirus (HCMV) gene prod- 44. Hmama, Z., R. Gabathuler, W. A. Jefferies, G. de Jong, and N. E. Reiner. 1998. ucts US2 and US11. J. Exp. Med. 188:497. Attenuation of HLA-DR expression by mononuclear phagocytes infected with 15. Ahn, K., A. Gruhler, B. Galocha, T. R. Jones, E. J. Wiertz, H. L. Ploegh, Mycobacterium tuberculosis is related to intracellular sequestration of immature P. A. Peterson, Y. Yang, and K. Fruh. 1997. The ER-luminal domain of the class II heterodimers. J. Immunol. 161:4882. HCMV glycoprotein US6 inhibits peptide translocation by TAP. Immunity 6:613. 45. Gruneberg, U., T. Rich, C. Roucard, S. M. van Ham, D. Charron, and ␣ 16. Hengel, H., J. O. Koopmann, T. Flohr, W. Muranyi, E. Goulmy, G. J. Hammerling, J. Trowsdale. 1997. Two widely used anti-DR monoclonal antibodies bind to an U. H. Koszinowski, and F. Momburg. 1997. A viral ER-resident glycoprotein inac- intracellular C-terminal epitope. Human Immunol. 53:34. tivates the MHC-encoded peptide transporter. Immunity 6:623. 46. Heinemann, D., P. J. Smith, and M. O. Symes. 1987. Expression of histocom- 17. Gilbert, M. J., S. R. Riddell, B. Plachter, and P. D. Greenberg. 1996. Cytomeg- patibility antigens and characterisation of mononuclear cell infiltrates in human alovirus selectively blocks antigen processing and presentation of its immediate- renal cell carcinomas. Br. J. Cancer 56:433. early gene product. Nature 383:720. 47. Kacani, L., I. Frank, M. Spruth, M. G. Schwendinger, B. Mullauer, G. M. Sprinzl, 18. Jonjic, S., I. Pavic, P. Lucin, D. Rukavina, and U. H. Koszinowski. 1990. Effi- F. Steindl, and M. P. Dierich. 1998. Dendritic cells transmit human immunode- cacious control of cytomegalovirus infection after long-term depletion of CD8ϩ ficiency virus type 1 to monocytes and monocyte-derived macrophages. J. Virol. T lymphocytes. J. Virol. 64:5457. 72:6671. 19. Jonjic, S., W. Mutter, F. Weiland, M. J. Reddehase, and U. H. Koszinowski. 48. Muczynski, K. A., S. K. Anderson, and D. Pious. 1998. Discoordinate surface 1989. Site-restricted persistent cytomegalovirus infection after selective long- expression of IFN-␥-induced HLA class II proteins in nonprofessional antigen- term depletion of CD4 T lymphocytes. J. Exp. Med. 169:1199. presenting cells with absence of DM and class II colocalization. J. Immunol. 20. Lucin, P., I. Pavic, B. Polic, S. Jonjic, and U. H. Koszinowski. 1992. ␥ interferon- 160:3207. dependent clearance of cytomegalovirus infection in salivary glands. J. Virol. 49. Carlsson, S. R., J. Roth, F. Piller, and M. Fukuda. 1988. Isolation and charac- 66:1977. terization of human lysosomal membrane glycoproteins, H-LAMP-1 and 21. Davignon, J.-L., P. Castanie, J. A. Yorke, N. Gautier, D. Clement, and H-LAMP-2: major sialoglycoproteins carrying polylactosaminoglycan. J. Biol. C. Davrinche. 1996. Anti-human cytomegalovirus activity of cytokines produced Chem. 263:18911. by CD4ϩ T-cell clones specifically activated by IE1 peptides in vitro. J. Virol. 50. Hammond, C., L. K. Denzin, M. Pan, J. M. Griffith, H. J. Geuze, and 70:2162. P. Cresswell. 1998. The tetraspan protein CD82 is a resident of MHC class II 22. Hengel, H., P. Lucin, S. Jonjic, T. Ruppert, and U. H. Koszinowski. 1994. Res- compartments where it associates with HLA-DR, -DM, and -DO molecules. toration of cytomegalovirus antigen presentation by ␥ interferon combats viral J. Immunol. 161:3282. escape. J. Virol. 68:289. 51. Smith, M. E. F., C. S. Holgate, J. M. S. Williamson, I. Grigor, P. Quirke, and 23. Lindsley, M. D., D. J. Torpey III, and C. R. Rinaldo, Jr. 1986. HLA-DR-restricted C. C. Bird. 1987. Major histocompatibility complex class II antigen expression in cytotoxicity of cytomegalovirus-infected monocytes mediated by Leu-3-positive B and T cell non-Hodgkin’s lymphoma. J. Clin. Pathol. 40:34. T cells. J. Immunol. 136:3045. 52. Heise, M. T., J. L. Pollock, A. O-Guin, M. L. Barkon, S. Bromley, and 24. Cresswell, P. 1996. Invariant chain structure and MHC class II function. Cell H. W. Virgin. 1998. Murine cytomegalovirus infection inhibits IFN-␥ induced 84:505. MHC class II expression on macrophages: the role of type I IFN. Virology 241: 25. Roche, P. A., M. S. Marks, and P. Cresswell. 1991. Formation of a nine-subunit 331. complex by HLA class II glycoproteins and the invariant chain. Nature 354:392. 53. Redpath, S., A. Angulo, N. R. J. Gascoigne, and P. Ghazal. 1999. Murine cyto- 26. Cresswell, P., and J. Howard. 1997. Antigen recognition. Curr. Opin. Immunol. megalovirus infection down-regulates MHC class II expression on macrophages 9:71. by induction of IL-10. J. Immunol. 162:6701. 27. Sedmak, D. D., A. M. Guglielmo, D. A. Knight, D. J. Birmingham, E. H. Huang, 54. Viville, S., J. Neefjes, V. Lotteau, A. Dierich, M. Lemeur, H. Ploegh, C. Benoist, and W. J. Waldman. 1994. Cytomegalovirus inhibits major histocompatibility and D. Mathis. 1993. Mice lacking the MHC class II-associated invariant chain. class II expression on infected endothelial cells. Am. J. Pathol. 144:683. Cell 72:635. 176 HCMV DISRUPTS CONSTITUTIVE MHC CLASS II

55. Pierre, P., and I. Mellman. 1998. Exploring the mechanisms of antigen processing 69. Wubbolts, R., and J. Neefjes. 1999. Intracellular transport and peptide loading of by cell fractionation. Curr. Opin. Immunol. 10:145. MHC class II molecules: regulation by chaperones and motors. Immunol. Rev. 56. Elliott, E. A., J. R. Drake, S. Amigorena, J. Elsemore, P. Webster, I. Mellman, 172:189. and R. A. Flavell. 1994. The invariant chain is required for intracellular transport 70. Bizario, J. C., F. A. Castro, R. N. Fernandes, A. D. Darniao, M. K. Olivera, and function of major histocompatibility complex class II molecules. J. Exp. Med. P. V. Palma, R. E. Larson, J. C. Voltarelli, and E. M. Espreafico. 2002. Myosin-V 179:681. colocalizes with MHC class II in blood mononuclear cells and is upregulated by 57. Anderson, H. A., and P. A. Roche. 1998. Phosphorylation regulates the delivery T-lymphocyte activation. J. Leukocyte Biol. 71:195. of MHC Class II invariant chain complexes to antigen processing compartments. 71. Barois, N., F. Forquet, and J. Davoust. 1998. Actin microfilaments control the J. Immunol. 160:4850. MHC class II antigen presentation pathway in B cells. J. Cell Sci. 111:1791. 58. Amigorena, S., P. Webster, J. Drake, J. Newcomb, P. Cresswell, and I. Mellman. 72. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen 1995. Invariant chain cleavage and peptide loading in major histocompatibility by cultured human dendritic cells is maintained by granulocyte/macrophage col- complex class II vesicles. J. Exp. Med. 181:1729. ony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis 59. Villadangos, J. A., R. J. Riese, C. Peters, H. A. Chapman, and H. L. Ploegh. 1997. factor ␣. J. Exp. Med. 179:1109. Degradation of mouse invariant chain: roles of cathepsins S and D and the in- 73. Pierre, P., S. J. Turley, E. Gatti, M. Hull, J. Meltzer, A. Mirza, K. Inaba, fluence of major histocompatibility complex polymorphism. J. Exp. Med. 186: R. M. Steinman, and I. Mellman. 1997. Developmental regulation of MHC class 549. II transport in mouse dendritic cells. Nature 388:787. 60. Pierre, P., and I. Mellman. 1998. Developmental regulation of invariant chain 74. Cella, M., A. Engerling, V. Pinet, J. Pieters, and A. Lanzavecchia. 1997. Inflam- proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell 93: matory stimuli induce accumulation of MHC class II complexes on dendritic 1135. cells. Nature 388:782. 61. Jones, T. R., L. K. Hanson, L. Sun, J. S. Slater, R. M. Stenberg, and 75. Winzler, C., P. Rovere, M. Rescigno, F. Granucci, G. Penna, L. Adorini, A. E. Campbell. 1995. Multiple independent loci within the human cytomegalo- V. S. Zimmermann, J. Davoust, and P. Ricciardi-Castagnoli. 1997. Maturation virus unique short region down-regulate expression of major histocompatibility stages of mouse dendritic cells in growth factor-dependent long-term cultures. complex class I heavy chains. J. Virol. 69:4830. J. Exp. Med. 185:317. 62. Riegler, S., H. Hebart, H. Einsele, P. Brossart, G. Jahn, and C. Sinzger. 2000. Monocyte-derived dendritic cells are permissive to the complete replicative cycle 76. Lewandowski, G. A., D. Lo, and F. E. Bloom. 1993. Interference with major of human cytomegalovirus. J. Gen. Virol. 81:393. histocompatibility complex class II-restricted antigen presentation in the brain by Downloaded from 63. Jahn, G., S. Stenglein, S. Riegler, H. Einsele, and C. Sinzger. 1999. Human herpes simplex virus type 1: a possible mechanism of evasion of the immune cytomegalovirus infection of immature dendritic cells and macrophages. Inter- response. Proc. Natl. Acad. Sci. USA 90:2005. virology 42:365. 77. Lewandowski, G., M. V. Hobbs, and F. E. Bloom. 1994. Alteration of intrace- 64. Raftery, M. J., M. Schwab, S. M. Eibert, Y. Samstag, H. Walczak, and rebral cytokine production in mice infected with herpes simplex virus types 1 and G. Schonrich. 2001. Targeting the function of mature dendritic cells by human 2. J. Neuroimmunol. 55:23. cytomegalovirus: a multilayered viral defense strategy. Immunity 15:997. 78. Arend, S. M., R. G. J. Westendorp, and F. P. Kroon. 1996. Rejection treatment 65. Odeberg, J., and C. Soderberg-Naucler. 2001. Reduced expression of HLA class and cytomegalovirus infection as risk factors for Pneumocystis carinii pneumonia

␤ in renal transplant recipients. Clin. Infect. Dis. 22:920. http://www.jimmunol.org/ II molecules and interleukin-10- and transforming growth factor 1-independent suppression of T-cell proliferation in human cytomegalovirus-infected macro- 79. George, M. J., D. R. Snydman, B. G. Werner, J. Grifﬁth, M. E. Falagas, phage cultures. J. Virol. 71:5174. N. N. Dougherty, and R. H. Rubin. 1997. The independent role of cytomegalo- 66. Spencer, J. V., K. M. Lockridge, P. A. Barry, G. Lin, M. Tsang, M. E. T. Penfold, virus as a risk factor for invasive fungal disease in orthotopic liver transplant and T. J. Schall. 2002. Potent immunosuppressive activities of cytomegalovirus- recipients: Boston Center for Liver Transplantation CMVIG-Study Group. encoded interleukin-10. J. Virol. 76:1285. Am. J. Med. 103:106. 67. Wubbolts, R., M. Fernandez-Borja, L. Oomen, D. Verwoerd, H. Janssen, 80. Husni, R. N., S. M. Gordon, D. L. Longworth, A. Arroliga, P. C. Stillwell, J. Calafat, A. Tulp, S. Dusseljee, and J. Neefjes. 1996. Direct vesicular transport R. K. Avery, J. R. Maurer, A. Metha, and T. Kirby. 1998. Cytomegalovirus of MHC class II molecules from lysosomal structures to the cell surface. J. Cell infection is a risk factor for invasive aspergillosis in lung transplant recipients. Biol. 135:611. Clin. Infect. Dis. 26:753. 68. Wubbolts, R., M. Fernandez-Borja, I. Jordens, E. Reits, S. Dusseljee, 81. Walter, E. A., P. D. Greenberg, M. J. Gilbert, R. J. Finch, K. S. Watanabe, C. Echeverri, R. B. Vallee, and J. Neefjes. 1999. Opposing motor activities of E. D. Thomas, and S. R. Riddell. 1995. Reconstitution of cellular immunity

dynein and kinesin determine retention and transport of MHC class II-containing against cytomegalovirus in recipients of allogeneic bone marrow by transfer of by guest on September 24, 2021 compartments. J. Cell Sci. 112:785. T-cell clones from the donor. N. Engl. J. Med. 333:1038.