Immune Evasion MHC Class II Processing Pathway for Herpes

Herpes Simplex Virus Type 1 Targets the MHC Class II Processing Pathway for Immune Evasion

This information is current as Jürgen Neumann, Anna Maria Eis-Hübinger and Norbert of October 2, 2021. Koch J Immunol 2003; 171:3075-3083; ; doi: 10.4049/jimmunol.171.6.3075 http://www.jimmunol.org/content/171/6/3075 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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Herpes Simplex Virus Type 1 Targets the MHC Class II Processing Pathway for Immune Evasion1

Ju¨rgen Neumann,* Anna Maria Eis-Hu¨binger,† and Norbert Koch2*

HSV type 1 (HSV-1) has evolved numerous strategies for modifying immune responses that protect against infection. Important targets of HSV-1 infection are the MHC-encoded peptide receptors. Previous studies have shown that a helper T cell response and Ab production play important roles in controlling HSV-1 infection. The reduced capacity of infected B cells to stimulate CD4؉ T cells is beneﬁcial for HSV-1 to evade immune defenses. We investigated the impact of HSV-1 infection on the MHCII processing pathway, which is critical to generate CD4؉ T cell help. HSV-1 infection targets the molecular coplayers of MHC class II processing, HLA-DR (DR), HLA-DM (DM), and invariant chain (Ii). HSV-1 infection strongly reduces expression of Ii, which impairs formation of SDS-resistant DR-peptide complexes. Residual activity of the MHC class II processing pathway is diminished by viral envelope glycoprotein B (gB). Binding of gB to DR competes with binding to Ii. In addition, we found gB associated with DM molecules. Both, gB-associated DR and DM heterodimers are exported from the endoplasmic reticulum, as indicated by Downloaded from carbohydrate maturation. Evaluation of DR, DM, and gB subcellular localization revealed abundant changes in intracellular distribution. DR-gB complexes are localized in subcellular vesicles and restrained from cell surface expression. The Journal of Immunology, 2003, 171: 3075–3083.

he immune system is highly efﬁcient in defending against heterodimers. B cells play a vital role in the humoral defense of

viral infection. The virus-host relationship, however, is HSV-1 infection and might also be a target for virus evasion strat- http://www.jimmunol.org/ T balanced by virus evasion strategies (1). Infected cells egies (5). The transfer of mAb against glycoprotein D to T cell display viral sequences that are bound to MHC receptors at the cell subset-depleted mice demonstrated that Abs provide protection surface and are recognized by T cells. CD8ϩ T cells detect viral against HSV-1-induced disease (6). To counteract the host immune peptides derived from the biosynthesis of infected cells. Hence, system, HSV-1 might influence the presentation of viral sequences biosynthesis of viral proteins is directly linked to formation of by MHCII polypeptides. MHC class I (MHCI)3-peptide complexes exposed on cell mem- Unlike MHCI molecules that use cytoplasmic degradation as a branes. HSV type 1 (HSV-1) infections elicit both cytotoxic and source for peptides, MHCII heterodimers acquire their antigenic Ab responses. One mechanism employed by the virus to prevent peptides in endosomal/lysosomal compartments. Maturation of cytotoxic responses to HSV-1 infection is early inhibition of MHCII molecules along their biosynthetic route involves degra- by guest on October 2, 2021 MHCI molecule transport to cell surfaces (2, 3). Binding of the dation and release of the associated invariant chain (Ii) and renders HSV-1 polypeptide ICP47 to the TAP transporter inhibits peptide the heterodimer susceptible to peptide acquisition (7, 8). A final translocation from the cytosol to the endoplasmic reticulum (ER) step in activation is achieved by the nonclassical MHCII polypep- lumen and prevents presentation of viral sequences by MHCI mol- tide DM. DM heterodimers do not appear to bind peptides (9). ecules. Such presentation is important to elicit a CD8ϩ T cell re- Their task is to release a fragment of Ii, which is lodged in the sponse (3, 4). In addition to a cytotoxic response, an Ab response MHCII groove (10, 11). In addition, DM molecules edit peptides and additional defense mechanisms, such as the release of IFNs, for stable MHCII complexes and for other structural factors (12). provide protection against virus dissemination and reinfection. HSV-1 has been shown to inhibit the ability of lymphoblastoid MHC class II (MHCII) is one key to developing a pathogen-spe- B cells to stimulate CD4ϩ T cells (13). By interfering with specific cific immune response. Generation of CD4ϩ T cell help for Ab steps in the biosynthetic MHCII processing pathway, HSV-1 production requires stimulation by virus peptide-loaded MHCII polypeptides might manipulate MHCII presentation. Recently, we discovered that the HSV-1 glycoprotein B (gB), transiently expressed in COS-7 cells, binds to HLA-DR molecules (14). *Section of Immunobiology, Institute for Molecular Physiology, University of Bonn, The impact of HSV-1 infection on the MHCII processing path- Bonn, Germany; and †Institute for Medical Microbiology and Immunology, Univer- sity of Bonn Medical Center, Bonn, Germany way and the role of the virus envelope protein gB was investigated Received for publication April 17, 2003. Accepted for publication July 14, 2003. in this study. We demonstrate that HSV-1 infection strongly de- creases the amount of Ii in B lymphoblastoid cells. gB binds to DR 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 and to DM heterodimers, thereby manipulating the MHCII pro- with 18 U.S.C. Section 1734 solely to indicate this fact. cessing pathway. Our results suggest that HSV-1 uses at least two 1 This work was supported by a Grant Ko 810/6-1 from the Deutsche distinct mechanisms to down-regulate a CD4 T cell response: 1) Forschungsgemeinschaft. interruption of MHCII Ag processing by reduction of Ii expression 2 Address correspondence and reprint requests to Dr. Norbert Koch, Universita¬t Bonn, and 2) interaction of gB with DM and DR polypeptides. Abteilung Immunbiologie, Institut fu¬r Molekulare Physiologie Ro¬merstrasse 164, 53117 Bonn, Germany. E-mail address: [email protected] 3 Abbreviations used in this paper: MHCI and II, MHC class I and class II; HSV-1, Materials and Methods HSV type 1; Ii, invariant chain; ER, endoplasmic reticulum; gB, glycoprotein B; Biochemicals and Abs NP40 NonidetP-40; EndoH, endoglycosidase H; PGNaseF, N-glycosidase F; CatB, cathepsin B; moi, multiplicity of infection; PVDF, polyvinylidene difluoride; HCMV, Bu-45 is a mouse mAb directed against the luminal domain of human Ii human CMV; LC, Langerhans cell. (15) and was used for immunoprecipitation. Immunoprecipitation of DR

FIGURE 1. MHC and HSV-1 protein expression by infected cells. A, JESTHOM cells were infected with HSV-1 for 24 h. Infected cells were MACS selected with mAb 2c/2 against gB, surface stained for HSV-1 proteins, and assayed by flow cytometry (upper panel). Uninfected cells incubated with anti-HSV-1 Abs are displayed as a dark curve. MHCII (middle panel) and MHCI (lower panel) surface molecules were stained with fluorescein-conjugated mAbs L243 and W6/32 (dark curve, uninfected cells; white curve, infected cells). B, Lysates from cells exposed to HSV-1 for 18, 40, and 64 h; MACS selected for gB and from uninfected cells were immunoblotted and stained for gB (mAb 2c/2), actin, and Ii (mAbs AC40 and Bu-43), DR␣ (mAb 1B5) MHCI H chain (mAb HC10), and DM␣ (mAb 5C1). The relative amount of Ii was estimated by densitometric scanning (data not shown). C, JESTHOM cells were infected with HSV-1. After 8 h, only a small fraction of the cells display gB on the cell surface. gB-expressing cells were separated by MACS. Cell lysates from infected and uninfected cells were immunoblotted and stained with mAbs for actin and Ii. D, JESTHOM cells were infected with HSV-1 for 72 h followed by MACS selection. Infected and uninfected cells were starved, metabolically labeled with [35S]methionine, and chased for up to 180 min. Ii was immunoprecipitated from cell lysates with mAb Bu-45. SDS-PAGE-separated immunoprecipitates were exposed to x-ray filmsfor4h(upper panels). The gel shown in the upper left panel in addition was exposed for 12 h (middle left). The intensity of Ii bands was densitometrically scanned (lower panels). The estimated t1/2 of Ii in infected and uninfected cells is indicated. was performed with mAb ISCR3 (16) while FITC-conjugated mAbs L243 class I was performed with FITC-conjugated mAb W6/32 (18), and stain- (17), Tu¬39-FITC (BD PharMingen, San Diego, CA), and I251 were used ing of cell surface-expressed HSV-1 proteins was achieved with antiserum for flow cytometry and fluorescence staining. Cell surface staining of HLA purchased from DAKO (Glostrup, Germany). HLA-DM and gB from The Journal of Immunology 3077

HSV-1 were immunoprecipitated with mAbs Map.DM1 (BD PharMingen) The reaction mixture was incubated for 16 h at 37¡C. Samples were ana- and 2c/2 (19). Western blot detection of HLA-DR, HLA-class I, Ii, gB, and lyzed by SDS-PAGE. actin was achieved with mAbs 1B5 (20), HC10 (21), Bu-43 (15), 10B7 (Virusys East Coast Biologics, North Berwick, USA), and AC40 (Sigma- Cell surface biotinylation Aldrich, Deisenhofen, Germany). DM was detected with Abs DM.K8 (DM␤, kindly provided by Dr. Moldenhauer DFKZ, Heidelberg, Germany) JESTHOM cells were infected for 24 h with HSV-1 at a moi ϭ 1 and were and 5C1 against DM␣ (22) N-glycosidase H and F were purchased from subsequently biotinylated using a standard protocol. In brief, 1 ϫ 107 cells New England Biolabs (Beverly, MA). Rabbit antiserum to cathepsin B was were suspended in 1 ml of biotinylation buffer (50 mM boric acid and 150 purchased from Calbiochem (Bad Soden, Germany). mM NaCl). Ten microliters of sulfosuccinimidyl-6-biotinamido-6-hexana-

midohexanoate (10 mg/ml in H2O; Pierce, Rockford, IL) was added and cDNA and vectors incubated for 15 min. The reaction was stopped by addition of 20 ␮lof100 ␣ ␤ mM NH4Cl. The samples were washed twice in ice-cold PBS and stored at The coding sequences of HLA-DR1, HLA-DM - and -chains, gB from Ϫ HSV-1 strain 17, and human Ii were cloned downstream of the CMV 70¡C. immediate early promoter in the vector pcDNA3.1 (Invitrogen, Karlsruhe, Germany) (14). Immunofluorescence microscopy Infection of cells COS-7 cells were transfected, seeded onto chamber slides (Nunc, Roskilde, Denmark) and cultured for 36 h. Cells were washed with PBS, fixed in COS-7 cells and the DR1 homozygote B lymphoma cell line JESTHOM PBS/4% paraformaldehyde, washed, and permeabilized with PBS/0.1% were cultured in DMEM supplemented with 0.4% glucose, 5% FCS, glu- Triton X-100 for 10 min. Subsequently, cells were washed and blocked tamine, penicillin/streptomycin, sodium pyruvate, and HEPES. For prop- with PBS/5% BSA for 1 h. After incubation with primary Ab overnight at agation of HSV-1 strain 17, which was used throughout this study, con- 4¡C, cells were washed, incubated with secondary Ab (goat anti-mouse- fluent Vero cell monolayers were infected at a multiplicity of infection Alexa Fluor 488, goat anti-mouse-Alexa Fluor 594, and goat anti-rabbit- (moi) of ϳ0.1 and serum concentration was lowered to 5%. Virus titration Alexa Fluor 488; Molecular Probes, Eugene, OR), diluted 1/400 in PBS/ Downloaded from was performed by the end point dilution method (23). 0.2% BSA for1hat37¡C, mounted with Mowiol (Sigma-Aldrich), and visualized by fluorescence microscopy (Axiophot; Zeiss, Oberkochen, Flow cytometry Germany). JESTHOM cells were washed twice in ice-cold PBS containing 2% FCS and 0.05% sodium azide. Rabbit anti-HSV-1 serum (DAKO) was added and cells were incubated for 30 min at 4 ¡C with FITC-conjugated goat Results anti-rabbit polyclonal Ab (Dianova, Hamburg, Germany). Staining of cell HSV-1 infection impairs expression of the MHCII-associated Ii http://www.jimmunol.org/ surface-expressed MHCI and MHCII was conducted with FITC-conju- To study the molecular impact of the HSV-1 polypeptide gB on the gated mAbs W6/32 and L243. After Ab addition the cells were incubated for 30 min at 4¡C. Cells were washed three times and analyzed with a BD MHCII processing pathway, the DR1 homozygous B lymphoblas- Biosciences FACScan unit (Mountain View, CA). toid cell line JESTHOM was infected with HSV-1. Since infection of B cells with HSV-1 is inefficient, infected cells were separated Magnetic bead separation of infected cells using Ab-bound magnetic beads. After 24 h of virus inoculation, JESTHOM cells were inoculated with HSV-1 (moi ϭ 1) and cultured for infected cells were sorted with gB-specific mAb. Flow cytometry 24, 36, 48, or 60 h. For magnetic bead separation of infected cells, mAb of surface-expressed HSV-1 proteins on separated cells showed ϫ 7 2c/2 (specific for gB) was used. Infected cells (1 10 ) were incubated that the vast majority of cells were infected (Fig. 1A, upper panel). with mAb 2c/2 for 30 min at 4¡C in a total volume of 100 ␮l. Cells were by guest on October 2, 2021 washed twice in ice-cold PBS/2% BSA and subsequently incubated with MHCII (HLA-DR) surface expression, with MHCI expression in- Ј anti-mouse-IgG2a/b F(ab )2-coated beads (Miltenyi Biotec, Bergisch Glad- cluded as a control, was monitored with FITC-conjugated mAbs bach, Germany). Cells were washed in PBS/2% BSA at 4¡C before transfer L243 and W6/32 (Fig. 1A, middle and lower panels). Infection to MACS columns (Miltenyi Biotec). Columns were washed three times with HSV-1 resulted in a moderate decrease in surface expression with PBS/2% BSA before cells were flushed out. of MHCII and MHCI molecules. It was reported that HSV-1 in- Transient transfection of cells fection retains MHCI molecules in the ER because of inhibition of COS-7 cells were transfected with the liposomal transfection reagent TAP-mediated peptide translocation (3). The small level of de- DOSPER (Roche, Mannheim, Germany). DNA mixed with DOSPER was crease of MHCI and MHCII surface expression, that we observed incubated for 20 min and added to cells. After 48 h, cells were harvested within the short time of HSV-1 infection, could be explained by and subjected to Western blot analysis or metabolic radiolabeling. the slow turnover rate of MHC molecules. For presentation of viral Metabolic radiolabeling, immunoprecipitation, SDS-PAGE and peptides however, the pool of MHC polypeptides delivered from Western blotting the biosynthesis of the cell is of particular importance. The long half-life of MHC molecules (24) may delay extensive ϫ 6 For metabolic labeling, 5 10 HSV-1-infected cells were starved for 45 reduction of surface class I and class II polypeptides in infected min in methionine-free RPMI 1640, followed by a 15-min pulse with 50 ␮Ci [35S]methionine. In some experiments, cells were recultured in me- cells. However, expression of MHCII-associated Ii, which exhibits dium containing 150 ␮g/ml nonradioactive methionine for up to 5 h. a half-life of only 2Ð3 h (25), could be affected by HSV-1 infec- For immunoprecipitation, cells were lysed in 0.5% Nonidet P-40 (NP40; tion. To evaluate the total amount of Ii, DR, and DM, gB-express- Sigma-Aldrich) containing the protease inhibitors aprotinin, PMSF, and ing cells were separated at 18, 40, or 64 h after HSV-1 infection. trypsin inhibitor (Sigma-Aldrich). Cell debris was removed by centrifuga- tion, and lysates were precleared by precipitation with CL4B-Sepharose The yield of gB-expressing cells was monitored by flow cytometry (Amersham Pharmacia Biotech, Piscataway, NJ). Supernatants were im- of infected cells. After separation, almost all cells stained positive munoprecipitated with 25 ␮l of 20-fold-concentrated hybridoma superna- for HSV-1 polypeptides (data not shown). Staining of dead cells tant and protein A-Sepharose. Protein A-bound immunoprecipitates were with propidium iodide revealed that the lytic activity of HSV-1 washed three times with 0.25% NP40 in TBS and subsequently separated increases with infection time at 64 h to ϳ20% of the cells (data not by SDS-PAGE and stained for Western blotting. For immunoblotting, cells were lysed in 0.5% NP40 buffer, electropho- shown). Cell lysates from infected and uninfected cells were sep- resed, and transferred to polyvinylidene difluoride (PVDF) membranes. arated by SDS-PAGE and analyzed by Western blotting (Fig. 1B). The PVDF membrane was blocked with RotiBlock (Roth, Karlsruhe, Ger- gB expression increases with infection time, suggesting continued many) and probed with Ag-specific primary Ab. Detection of primary Ab gB synthesis. Staining with mAb Bu-43 against the Ii revealed a binding was conducted with HRP-coupled Ab and ECL Western blotting ϳ reagent (Amersham Pharmacia Biotech). Immunoisolates were deglycosy- dramatic reduction of Ii in infected cells, down to 14% after lated with endoglycosidase H (EndoH) and N-glycosidase F (PGNaseF). 64 h, when compared with uninfected cells. Immunostaining for Digestion was performed in the buffer recommended by the manufacturer. actin confirmed that equivalent numbers of cells were assayed. The 3078 MHC CLASS II IS TARGETED BY HSV-1 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. (Figure legend continues) The Journal of Immunology 3079 total amount of DR␣, MHCI H chain, and DM␣ remains unaf- which stained with DR␣-specific mAb 1B5. SDS-stable DR com- fected in infected cells. A significant decrease in Ii expression was plexes were strongly reduced in infected cells compared with un- detected as early as 18 h after infection. However, Ii expression infected cells (Fig. 2A, lanes 1 and 3). In lysates from infected was not reduced 8 h after infection (Fig. 1C). Thus, the vast ma- cells, most DR␣ appears as monomeric polypeptide, while in un- jority of Ii vanishes between 8 and 18 h of infection. Since Ii plays infected cells, about half of the DR␣ is found in SDS-stable com- an important role in Ag processing (26), the loss of Ii expression plexes. After boiling the samples, which causes dissociation of the may contribute to the decline of MHCII Ag presentation by HSV- SDS-resistant DR complexes, the intensity of DR␣ bands in in- 1-infected cells. The small amount of Ii detected in infected cells fected and uninfected cells was similar (Fig. 2A, lanes 2 and 4). by immunoblotting (Fig. 1B) suggests that Ii is rapidly degraded, Western blots for actin were used to confirm that similar amounts perhaps by a viral proteinase. The stability of Ii was investigated of infected and uninfected cell lysates were analyzed (Fig. 2A, by a pulse-chase experiment in infected and uninfected cells (Fig. lanes 5 and 6). The result in Fig. 2A suggests that a large amount 1D). Cells were pulse-labeled for 15 min with [35S]methionine and of the DR polypeptide in HSV-1-infected cells is not associated chased for the times indicated. The low rate of Ii synthesis in with peptides, at least not as heat-labile DR complexes. infected cells (Fig. 1D, upper left panel) was opposed to synthesis in uninfected cells (Fig. 1D, upper right panel). Compared with the In HSV-1-infected B cells gB associates with DR and with DM half-life of Ii in uninfected cells after 4-h exposure (Fig. 1D, upper heterodimers right panel), infected cells exposure for 12 h (Fig. 1D, middle HSV-1-infected B cells were lysed with NP40 and immunopre- panel) showed rapid Ii degradation, but the decay is not acceler- cipitated for gB. Immunoblotting was conducted with Abs against ated upon HSV-1 infection as can be judged from a densitometric DR␣ and against gB. Fig. 2B (left panel) shows that DR was coiso- Downloaded from scan (Fig. 1D, lower panel). These results suggest that HSV-1 lated with gB (lane 1). Expression of gB and DR is demonstrated infection reduces Ii biosynthesis and that physiological degrada- by immunoblotting of cell lysates (Fig. 2B, left panel, lanes 3 and tion leads to low levels of Ii expression. The decline of biosyn- 4). It appeared possible that gB might also bind to the MHCII-like thesis by HSV-1 infection appears to be nonspecific and also con- molecule DM. DM, a peptide binding editor, is required to render cerns MHCI and MHCII polypeptides (27), which we confirmed the MHCII groove susceptible for peptide binding. To explore a by immunoprecipitation of [35S]methionine-labeled MHCII mol- possible interaction between gB and DM under physiological con- ecules from HSV-1-infected cells (data not shown). To eliminate ditions, B lymphoblastoid cells were infected with HSV-1 or were http://www.jimmunol.org/ the activity of residual MHCII molecules derived from biosynthe- left uninfected, lysed, and immunoprecipitated using mAb against sis, HSV-1 may have evolved a strategy to inhibit loading of viral gB (Fig. 2B, right panel). We assumed that the potential binding of peptides to newly synthesized MHCII polypeptides to abolish Ag gB to DM was similar to the binding of Ii to DM. Therefore, since presentation. Therefore, it is possible that HSV-1 in addition tar- detection of Ii binding to DM depends on mild detergent, cells gets the MHCII processing pathway. were lysed with digitonin. Immunoprecipitates (Fig. 2B, right panel, lanes 1 and 2) and lysates (lanes 3 and 4) were separated by Peptide loading to MHCII heterodimers is impaired in SDS-PAGE followed by Western blotting with Abs directed to gB HSV-1-infected cells ␣

and DM . To exclude that the interaction of DM to gB occurs via by guest on October 2, 2021 Ag-specific T cell stimulation is sensitive to a low number of DR molecules, we tested binding in the absence of DR. COS-7 MHCII-presented epitopes. The level of biosynthesis in HSV-1- cells were transfected with gB and DM, lysed with digitonin, and infected cells might permit Ag processing and support presentation immunoprecipitated for gB. Again, DM␣ was detected by Western by MHCII molecules at levels sufficient to stimulate CD4ϩ T cells. blot, confirming association of gB with DM (data not shown). It was unclear whether generation of peptide MHCII complexes is Lysates separated in Fig. 2B, right panel, lanes 3 and 4, show affected during HSV-1 infection. To evaluate the ability of infected expression of DM and verify expression of gB in infected cells. B lymphoblastoid cells to present Ag, the level of SDS-stable Lane 1 shows that DM␣ was coprecipitated with gB, indicating MHCII complexes, which represent a fraction of peptide-loaded that gB binds to DM molecules. Binding of gB to DM was not MHCII heterodimers, was examined in lysates from infected and detected after mixing of cell lysates from gB- or DM-transfected uninfected cells by immunoblotting (Fig. 2A). Nonboiled samples COS-7 cells and subsequent immunoprecipitation of DM or gB resolved into SDS-resistant DR complexes and monomeric bands, (Fig. 2C, lanes 2 and 4). This result confirms that the association

FIGURE 2. MHCII peptide loading and binding of gB to DR and DM heterodimers. A, HSV-1-infected JESTHOM cells were propagated for 64 h and separated with gB mAb by MACS. Lysates from infected and uninfected cells were divided into two aliquots. One sample was boiled in SDS sample buffer (b), the other probe was incubated for1hatroom temperature in SDS sample buffer (nb), followed by SDS-PAGE and immunoblotting with mAb 1B5 (DR␣)(lanes 1Ð4). Cell lysates were also immunoblotted for actin (lanes 5 and 6). The positions of SDS-resistant DR complexes and actin and DR␣ bands are indicated on the right. B, JESTHOM cells were infected with HSV-1 for 48 h. gB-expressing cells were MACS selected with mAb 2c/2 (gB). Infected and uninfected cells were lysed with 0.5% NP40 (left panel) or with 0.5% digitonin (right panel) and gB was immunoprecipitated (IP) with mAb 10B7 (lanes 1 and 2). Cell lysates were separated in lanes 3 and 4. After immunoblotting, the polypeptides were stained with a pool of mAbs against gB (10B7) and mAb against DR␣ (1B5, left panel) or with mAb against gB (10B7) and mAb against DM␣ (5C1, right panel). The positions of gB, DR␣, and DM␣ bands are indicated on the right and Ig H and L chains on the left. C, COS-7 cells were transiently transfected with DM or with gB-encoding cDNAs. Transfected and untransfected cells were lysed with 0.5% digitonin. Lysates from DM- and from gB-transfected cells were pooled, incubated for 30 min, and immunoprecipitated against DM (mAb MaP.DM1) or gB (mAb 2c/2) (lanes 2 and 4). Lysates from untransfected cells were immunoprecipitated as a control (lanes 1 and 3). Immunoblotting was conducted with a pool of mAbs against DM␣ (mAb 5C1) and against gB (mAb 10B7). Lysates of gB- and DM-transfected cells were separated in lanes 6Ð8. Lysate from untransfected cells is shown in lane 5. Lysates were immunoblotted using Abs against gB (lane 6), DM␣ (lane 7), and DM␤ (mAb DMK.8) (lane 8). The positions of gB, DM␣, and DM␤ bands are indicated on the right and H and L Ig chains on the left. D, COS-7 cells were transiently transfected with DR1-, gB-, and Ii-encoding cDNAs. Transfected and untransfected cells were lysed and immunoprecipitated using Abs against Ii (mAb Bu-45) and gB (mAb 2c/2) (lanes 1Ð4). The immunoblotted polypeptides were stained with a pool of Abs against Ii (Bu-43) and gB (10B7). Cell lysate was separated in lanes 5Ð7 and immunoblotted with mAbs against Ii (lane 5), DR␣ (lane 6), and DR␤ (lane 7). The positions of gB, as well as Ig H and L chains are indicated on the left and DR␣, Ii, and DR␤ on the right. 3080 MHC CLASS II IS TARGETED BY HSV-1

FIGURE 3. Intracellular transport and localization of DR and DM in gB-expressing cells. A and B, glycan maturation of gB-associated DM and DR glycopro- teins. JESTHOM cells were infected with HSV-1 for 48 h and separated by MACS with mAb against gB. Cells were lysed in 0.5% NP40 (A) or 0.5% digitonin (B). Ly- sates from infected cells were subjected to immunoprecipitation (IP) with mAb 10B7 against gB. The immunocomplexes and cell lysates from uninfected cells were digested with EndoH (EH) for 16 h at 37¡C(A, lanes 2 and 5; B, lanes 2 and

4), digested with PNGaseF (PF) for 16 h at 37¡C(A, lanes 3 and 6; B, lane 5), or left untreated (A, lanes 1 and 4; B, lanes 1 and 3). Staining of immunoblotted Downloaded from polypeptides was conducted with mAb 1B5 against DR␣ (A) or mAb 5C1 against DM␣ (B). The positions of DR␣,DM␣, gB, and Ig H and L chains are indicated. C, DR, DM, and gB are localized in vesicular cytosolic compartments by fluorescence microscopy. COS-7 cells were http://www.jimmunol.org/ transfected with DR1, gB, DM, or Ii as indicated. After 36 h, the cells were fixed, permeabilized, and incubated with mouse mAbs to DR (I251), Ii (Bu-43 and Bu- 45), DM (Map.DM1 and 5C1), and gB (2c/2), as indicated, followed by incubation with a second fluorescein-conjugated Ab. For double staining, gB- and DR-

transfected COS-7 cells were first incu- by guest on October 2, 2021 bated with gB mAb 2c/2, which was la- beledwithAlexaFluor594-conjugatedanti- mouse Ab, followed by incubation with FITC-conjugated DR Ab Tu¬-39 (IV). The nuclei were counterstained with bis-benzimide. D, Costaining of DR and DM with the lysosomal/endosomal marker CatB. COS-7 cells were transfected with DR/Ii (I), with DR/gB (II), with DM (III), and with DM/gB (IV). The first line (I and II) was stained for DR and III and IV were stained for DM. The middle line was stained for CatB. In the third line, staining of lines 1 and 2 was merged. Colocaliza- tion is indicated by yellow staining. E, gB-associated DR is detained from the cell surface. COS-7 cells were transfected with gB, DR1, or Ii as indicated. After 36 h, the cells were biotinylated and subjected to immunoprecipitation with a mAb to gB (2c/2) (lanes 1 and 3) or with a mAb to DR (I251) (lane 2). Mock-transfected cells were immunoprecipitated for MHCI (mAb W6/32) or for actin (mAb AC40). The immunocomplexes were separated by SDS-PAGE, transferred to a PVDF membrane and incubated with streptavidin-peroxidase followed by ECL detection. The Journal of Immunology 3081 of gB with DM occurs in infected cells. Immunoblotting of the tained for DR (Fig. 3II, middle). Expression of DM or DM and gB lysates indicates expression of either gB or DM␣␤ chains (Fig. 2C, (Fig. 3III) resulted in a subcellular distribution similar to DR/Ii in lanes 6Ð8). the first and second panel, except that vesicular staining of DM throughout the cytoplasm did not depend on Ii. Coexpression of gB HSV-1-encoded gB competes with Ii for binding to MHCII and DM altered the subcellular distribution of the stained polypep- heterodimers tides (Fig. 3III, middle and bottom). To demonstrate colocalization Recently, we found that the HSV-1 envelope protein gB contains of DR and gB, cells were stained with FITC anti-DR and anti-gB a sequence motif that is also contained in Ii (14). It was possible (red; IV, top and middle). Merging of the red and green stains (IV, that Ii and gB compete for binding to MHCII molecules. Alterna- bottom) demonstrated colocalization of DR and gB in subcellular tively, Ii and gB may bind to the same DR heterodimer. To ex- vesicles. Expression of DR in gB-expressing cells resulted in a amine interactions among Ii, gB, and DR, COS-7 cells were trans- more distinct vesicular label than DR staining in the absence of Ii. fected with the corresponding cDNAs. Fig. 2D shows This result is consistent with our finding that the carbohydrate of immunoprecipitates and cell lysates separated by SDS-PAGE. Im- DR␣ acquires resistance to EndoH treatment, indicating migration munoblotting of the lysates indicates that Ii, DR␣, and DR␤ are of DR to Golgi compartments. expressed (Fig. 2D, lanes 5Ð7). Immunoprecipitates of Ii and gB To demonstrate intracellular localization of gB and MHCII mol- from transfected and nontransfected COS-7 cells were separated in ecules, we used cathepsin B (CatB) as a lysosomal/endosomal Fig. 2D, lanes 1Ð4. Western blotting of the SDS-PAGE separated marker (Fig. 3D). Coexpression of DR with Ii demonstrates ex- polypeptides revealed that Ii and gB were independently isolated pression of DR in CatB-containing compartments (yellow staining, (Fig. 2D, lanes 2 and 4). Ii and gB do not coisolate through binding I, bottom). The colocalization of DR with CatB is strongly reduced to DR. Thus, there are two DR populations, one composed of Ii in the presence of gB (compare yellow staining of I with II, both Downloaded from and DR, and one with DR and gB. However, there are no DR bottom). Similar results were obtained when DM and gB were complexes containing both gB and Ii. One can thus conclude that coexpressed. DM exhibits a lysosomal/endosomal distribution, a decrease of Ii and an increase in gB expression, that was ob- which in contrast to DR is achieved in the absence of Ii (yellow served upon HSV-1 infection, out-competes Ii in newly synthe- staining, III, bottom). This can be explained by an endosomal sort- sized DR complexes by gB. ing signal present on the DM␤ chain. The endosomal localization of DM is strongly altered when coexpressed with gB (IV, bottom). http://www.jimmunol.org/ gB migrates with associated DR or DM polypeptides from the Some coexpression of DM and CatB is still detected in the pres- ER to Golgi compartments ence of gB. However, a high amount of DM is intracellularly lo- The fate of gB-associated DR and DM after biosynthesis remains calized, different from lysosomal/endosomal staining (red vs yel- unclear. It has been found that HSV-1 infection retains MHCI low staining, IV, bottom). molecules in the ER and prevents intracellular transport and pre- In addition, gB-, DR-, and Ii-transfected COS-7 cells and HSV- sentation of peptides at cell surfaces (28). We examined whether 1-infected B lymphoblastoid cells were surface biotinylated. Sur- gB-associated DR and DM molecules are exported from the ER. face-labeled gB was immunoprecipitated from DR/gB-transfected Both DM and DR ␣-chains contain two N-linked carbohydrates. In COS-7 cells (Fig. 3E, lane 3). No surface-labeled DR coprecipi- by guest on October 2, 2021 uninfected cells, one of the two glycans of each polypeptide is tated with gB is visible in lane 3. Expression of DR in gB/DR- converted in the Golgi complex from an EndoH-sensitive to an transfected cell lysates was confirmed by Western blotting (data EndoH-resistant form, whereas the second glycan chain remains not shown). The position of SDS-PAGE-separated gB and DR EndoH sensitive (Fig. 3A, lane 5 and B, lane 4). DR and DM bands is shown in lanes 1 and 2. Immunoprecipitation of endog- molecules coprecipitated with gB also show resistance of one car- enously expressed and surface-biotinylated MHCI (lane 4) did not bohydrate chain to EndoH treatment (Fig. 3, A and B, lane 2). exhibit association with gB. Precipitation of small amounts of actin PNGaseF treatment of cell lysates and immunoprecipitated DR (lane 5) revealed that a low quantity of intracellular protein was molecules remove both glycan chains from DR␣ and DM␣ (Fig. labeled with biotin. A long exposure of the gel exhibits small 3A, lanes 3 and 6, and B, lane 5). The resistance of one of the amounts of DR bands in lane 3 (data not shown). Consistent with glycan chains to EndoH treatment indicates that gB-associated DM labeling of small amounts of actin, the appearance of these DR and DR polypeptides travel from the ER to Golgi compartments, bands can be explained by biotinylation of intracellular proteins where the carbohydrates are modified. This is similar to migration that were derived from some death cells. The result in Fig. 3E of DM and DR molecules in uninfected cells. suggests that gB-associated DR molecules are not expressed on the To assess subcellular localization of DR and DM in gB-express- cell surface. ing cells, COS-7 cells were transfected with cDNAs encoding DR, DM, gB, and as a control, Ii. Cells were seeded on chamber slides, Discussion fixed, and permeabilized before staining. Fig. 3C shows immuno- Recent research indicates that MHCII molecules and the Ag pro- staining of DR-, gB-, or Ii-expressing COS-7 cells. The nuclei cessing pathway are targets for virus evasion strategies. The HIV- were counterstained with bis-benzimide. Cells solely expressing encoded Nef protein inhibits cell surface expression of MHCII DR molecules (Fig. 3I, top) show staining of the perinuclear mem- molecules and presentation of peptides (29). Presumably, Nef af- brane and polarized distribution at intracellular membranes. This fects intracellular trafficking of MHCII polypeptides. The herpes pattern is characteristic of ER labeling as we showed by costaining virus human CMV (HCMV) impairs stability of DR and DM mol- for the ER marker protein disulfide-isomerase (data not shown). ecules and down-modulates DR surface expression during latent Upon coexpression of DR with Ii, staining for both DR and Ii infection (30, 31) In mice, it was demonstrated that the HCMV polypeptides is present in small vesicles distributed uniformly protein US2 binds to MHCII ␣␤ Ii complexes and blocks protein throughout the cytoplasm (Fig. 3I, middle and bottom). gB shows trafficking. US2 promotes degradation of DR␣ and DM␤ chains. In a staining pattern similar to that of DR coexpressed with Ii (Fig. contrast to US2, HSV-1 encoded gB binds only to Ii-free MHCII 3II, top). However, when coexpressed with DR, the distribution of heterodimers. It was reported, however, that HCMV-derived US3 gB vesicular staining is strongly reduced and does not extend to competes with Ii for binding to MHCII molecules (32). The bind- the cell membrane (Fig. 3II, bottom). A similar staining was ob- ing site of US3 to MHCII has not yet been identified. As shown in 3082 MHC CLASS II IS TARGETED BY HSV-1

HSV-1-infected cells, reduction of Ii levels is a way for the virus affecting the biosynthetic route of DR molecules, however, the aim to limit Ag processing. Similar observations have been made with of the virus is achieved: presentation of viral peptides to CD4ϩ T HIV-2-infected cells. The HIV-2 Vpx polypeptide interacts with Ii, cells is impaired. thereby reducing the level of Ii (33). gB contains a DR1-binding motif at the N terminus, which is of In a recent study, the reduced capacity of infected B lympho- interest, because gB is a target for CD4ϩ T cells (42). The DR1- blastoid cells to stimulate T cells was demonstrated (13). T cells binding sequence is flanked by a proline/lysine-rich sequence that specific for HSV-1-encoded gB and gD polypeptides were used in is also contained in Ii. The DR1-binding sequence and the proline/ this study. HSV-1-infected B lymphoblastoid cells used as APCs lysine-rich sequence of gB resemble the MHCII-binding sequence showed impaired stimulation of the gB- and gD-specific CD4ϩ T of Ii (14). Similar to the binding of gB to MHCII, a superantigen cells. Since only the infected B cells present the Ag the authors from mouse mammary tumor virus imitates the interaction be- provide evidence that HSV-1 inhibits the capacity of lymphoblas- tween Ii and MHCII heterodimers (43). toid B cells to activate Ag-specific T cells. It can be concluded that binding of gB to MHCII heterodimers It has been previously reported that HSV-1 infection impairs pro- inhibits peptide loading and prevents presentation of viral peptides. tein biosynthesis. Soon after infection the HSV-1-derived vhs protein, HSV-1-encoded gB is an envelope protein required for infection (44). which possesses RNase activity, induces nonspecific mRNA degra- gB attaches to negatively charged heparan sulfate moieties and pro- dation (34). In addition, viral protein ICP34.5 blocks protein synthesis motes fusion of the viral envelope with the cell membrane, followed by dephosphorylating the ␣ subunit of translation factor 2 (35). Re- by entry of virions into cells. The finding within this study that DR duced MHC polypeptide synthesis in HSV-1-infected glioblastoma expression changes the intracellular distribution of gB may have con- cells was demonstrated (27). Glioblastoma cells show some decrease sequences for virus particle production. gB polypeptides engaged in in MHCII cell surface expression although the total amount of MHCII DR binding may not be available to serve as virus envelope proteins. Downloaded from is constant. This result is consistent with our observation that HSV-1 Therefore, DR expression could reduce HSV-1 replication in APCs. infection does not alter total MHCII expression in B lymphoid cells. The 874-aa sequences of gB from various HSV-1 strains show high The data of Trgovcich et al. (27) further suggest that MHCII cell homology with a few patches of polymorphism. Two gB polymorphic surface expression is controlled by an unknown mechanism through residues are located in the PKPPKP sequence, the site where gB binds ␥ the HSV-1 genes UL 41 and 1 34.5. to MHCII heterodimers. These sequence variations can be related to HSV-1 is an important human pathogen that infects epithelial cells a mouse infection model. It has been reported that infection with the http://www.jimmunol.org/ and sensory ganglia in particular, but also infects other cell types with HSV-1 strain ANGpath is lethal to mice, whereas strain HSZP is varying efficiencies. After an acute episode, the virus retracts to the nonpathogenic (45, 46). Through transfer and mapping studies, this neural ganglia and remains in a latent state. HSV-1 has developed pathogenic phenotype could be attributed to the gB molecule (45, 47). multiple strategies to escape and persist within the host immune sys- The gB sequence of ANGpath is highly similar to HSV-1 strain 17, tem. During primary infection and numerous cycles of reactivation, which was used in this study. In contrast, HSZP and other nonpatho- HSV-1-encoded proteins modify immune responses to virus-infected genic HSV-1 strains show four mutations at the N terminus of gB cells. Virus evasion strategies also affect functional recognition of between aa residues 59 and 79 (48). Two residues, Pro77 and Lys79, APCs. HSV-1 infection blocks maturation of dendritic cells by inhib- of ANGpath were mutated in the nonpathogenic strains. These resi- by guest on October 2, 2021 iting the signaling pathway, which results in a reduced capacity to dues are also located in the MHCII binding site PKP77PK79PofgB stimulate allogeneic T cells (36). It is interesting to note that in the from strain 17 (49). The binding properties of gB to MHCII molecules absence of CD8ϩ T cells, a vaccine-induced immunity to HSV-1 could be related to the pathogenic phenotype of HSV-1 strain AN- infection can be achieved (37). In mice, the ability to control HSV-1 Gpath. Moreover, infection of mice with HSV-1 strains KOS or F infection is attributed to helper T cells. Susceptibility of CD4 T cell- results in a latent infection only with strain F, whereas spreading of deficient mice to HSV-1 infection challenges the primary importance the KOS strain is not controlled (50). The MHCII biosynthetic path- of this T cell subset and emphasizes the significance of Abs in me- way in HSV-1-infected cells appears to be interrupted in cells infected diating protection from HSV (38, 39), while the importance of anti- with the KOS strain. As shown in this report, gB efficiently interacts HSV-1 Abs in humans is less clear. Ab-producing B cells might be a with MHCII molecules in HSV-1-infected cells. A mutation of Pro80 potential virus target of HSV-1. in gB (strain F) to Thr80 (strain KOS), which is present in a MHCII- Down-regulation of MHCII and Ii biosynthesis, such as that binding sequence, could account for the variant interaction to mouse observed upon HSV-1 infection of B lymphoblastoid cells, shows Ia molecules in cells infected with strains KOS and F. some similarity to the maturation of Langerhans cells (LC). LC HSV-1 infection is endemic within the human population. Many internalize Ags that penetrate skin lesions and subsequently move individuals carry the virus without any apparent disease. In con- to lymph nodes, where they present antigenic epitopes to T cells. trast, others suffer from frequent virus reactivation and recurrent Under experimental conditions, LC display a differentiation pat- disease. One may assume that susceptibility to HSV-1 reactivation tern that resembles their in vivo phenotype as professional APCs is linked to a partial deficient immunity. It is tempting to speculate (40). LC isolated from skin and cultured for several days change that HLA polymorphism is associated with immunity to HSV-1 their phenotype. Upon tissue extraction of LC, biosynthesis of infection. The molecular basis for HSV-1-related immunity could MHCII and Ii is arrested. The ability to process Ag declines with involve interaction of the virus envelope protein gB with polymor- the decay of Ii. In the absence of Ii, degradation of mouse H2-M phic MHCII molecules. In early studies, natural resistance to her- is increased (41). Mature LC express MHCII molecules that have pes virus infection was linked to MHC haplotypes by examining acquired their peptides as immature LC. Our results suggest that H2-congenic mouse strains (51). There is evidence, that the human HSV-1 employs a similar but distinct mechanism to modulate T cell response to gB is influenced by MHC, but only limited MHCII Ag presentation. In HSV-1-infected cells, the MHCII pro- experimental data on HLA typing and HSV-1 infection are avail- cessing pathway is blocked because Ii vanishes. In addition, gB able (52, 53). Investigation of the genetic basis of HSV-1 immu- binds to DM and possibly separates the peptide editor from the nity might be an interesting topic for future studies. MHCII pathway. The small amount of SDS-resistant DR complexes detected in infected cells suggests that peptides loaded onto Acknowledgments MHCII heterodimers before infection can still be presented. By We thank Dr. G. Moldenhauer for providing Abs. The Journal of Immunology 3083

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