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The Journal of Immunology, 2003, 171: 3075-3083.
Copyright © 2003 by The American Association of Immunologists

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

Jürgen Neumann*, Anna Maria Eis-Hübinger{dagger} and Norbert Koch2,*

* Section of Immunobiology, Institute for Molecular Physiology, University of Bonn, Bonn, Germany; and {dagger} Institute for Medical Microbiology and Immunology, University of Bonn Medical Center, Bonn, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The immune system is highly efficient in defending against viral infection. The virus-host relationship, however, is balanced by virus evasion strategies (1). Infected cells display viral sequences that are bound to MHC receptors at the cell surface and are recognized by T cells. CD8+ T cells detect viral peptides derived from the biosynthesis of infected cells. Hence, biosynthesis of viral proteins is directly linked to formation of MHC class I (MHCI) 3-peptide complexes exposed on cell membranes. HSV type 1 (HSV-1) infections elicit both cytotoxic and Ab responses. One mechanism employed by the virus to prevent cytotoxic responses to HSV-1 infection is early inhibition of MHCI molecule transport to cell surfaces (2, 3). Binding of the HSV-1 polypeptide ICP47 to the TAP transporter inhibits peptide translocation from the cytosol to the endoplasmic reticulum (ER) lumen and prevents presentation of viral sequences by MHCI molecules. Such presentation is important to elicit a CD8+ T cell response (3, 4). In addition to a cytotoxic response, an Ab response and additional defense mechanisms, such as the release of IFNs, provide protection against virus dissemination and reinfection. MHC class II (MHCII) is one key to developing a pathogen-specific immune response. Generation of CD4+ T cell help for Ab production requires stimulation by virus peptide-loaded MHCII heterodimers. B cells play a vital role in the humoral defense of HSV-1 infection and might also be a target for virus evasion strategies (5). The transfer of mAb against glycoprotein D to T cell subset-depleted mice demonstrated that Abs provide protection against HSV-1-induced disease (6). To counteract the host immune system, HSV-1 might influence the presentation of viral sequences by MHCII polypeptides.

Unlike MHCI molecules that use cytoplasmic degradation as a source for peptides, MHCII heterodimers acquire their antigenic peptides in endosomal/lysosomal compartments. Maturation of MHCII molecules along their biosynthetic route involves degradation and release of the associated invariant chain (Ii) and renders the heterodimer susceptible to peptide acquisition (7, 8). A final step in activation is achieved by the nonclassical MHCII polypeptide DM. DM heterodimers do not appear to bind peptides (9). Their task is to release a fragment of Ii, which is lodged in the MHCII groove (10, 11). In addition, DM molecules edit peptides for stable MHCII complexes and for other structural factors (12).

HSV-1 has been shown to inhibit the ability of lymphoblastoid B cells to stimulate CD4+ T cells (13). By interfering with specific steps in the biosynthetic MHCII processing pathway, HSV-1 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).

The impact of HSV-1 infection on the MHCII processing pathway and the role of the virus envelope protein gB was investigated in this study. We demonstrate that HSV-1 infection strongly decreases the amount of Ii in B lymphoblastoid cells. gB binds to DR and to DM heterodimers, thereby manipulating the MHCII processing pathway. Our results suggest that HSV-1 uses at least two distinct mechanisms to down-regulate a CD4 T cell response: 1) interruption of MHCII Ag processing by reduction of Ii expression and 2) interaction of gB with DM and DR polypeptides.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Biochemicals and Abs

Bu-45 is a mouse mAb directed against the luminal domain of human Ii (15) and was used for immunoprecipitation. Immunoprecipitation of DR was performed with mAb ISCR3 (16) while FITC-conjugated mAbs L243 (17), Tü39-FITC (BD PharMingen, San Diego, CA), and I251 were used for flow cytometry and fluorescence staining. Cell surface staining of HLA class I was performed with FITC-conjugated mAb W6/32 (18), and staining of cell surface-expressed HSV-1 proteins was achieved with antiserum purchased from DAKO (Glostrup, Germany). HLA-DM and gB from HSV-1 were immunoprecipitated with mAbs Map.DM1 (BD PharMingen) and 2c/2 (19). Western blot detection of HLA-DR, HLA-class I, Ii, gB, and actin was achieved with mAbs 1B5 (20), HC10 (21), Bu-43 (15), 10B7 (Virusys East Coast Biologics, North Berwick, USA), and AC40 (Sigma-Aldrich, Deisenhofen, Germany). DM was detected with Abs DM.K8 (DM{beta}, kindly provided by Dr. Moldenhauer DFKZ, Heidelberg, Germany) and 5C1 against DM{alpha} (22) N-glycosidase H and F were purchased from New England Biolabs (Beverly, MA). Rabbit antiserum to cathepsin B was purchased from Calbiochem (Bad Soden, Germany).

cDNA and vectors

The coding sequences of HLA-DR1, HLA-DM {alpha}- and {beta}-chains, gB from HSV-1 strain 17, and human Ii were cloned downstream of the CMV immediate early promoter in the vector pcDNA3.1 (Invitrogen, Karlsruhe, Germany) (14).

Infection of cells

COS-7 cells and the DR1 homozygote B lymphoma cell line JESTHOM were cultured in DMEM supplemented with 0.4% glucose, 5% FCS, glutamine, penicillin/streptomycin, sodium pyruvate, and HEPES. For propagation of HSV-1 strain 17, which was used throughout this study, confluent Vero cell monolayers were infected at a multiplicity of infection (moi) of ~0.1 and serum concentration was lowered to 5%. Virus titration was performed by the end point dilution method (23).

Flow cytometry

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 anti-rabbit polyclonal Ab (Dianova, Hamburg, Germany). Staining of cell surface-expressed MHCI and MHCII was conducted with FITC-conjugated 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 Biosciences FACScan unit (Mountain View, CA).

Magnetic bead separation of infected cells

JESTHOM cells were inoculated with HSV-1 (moi = 1) and cultured for 24, 36, 48, or 60 h. For magnetic bead separation of infected cells, mAb 2c/2 (specific for gB) was used. Infected cells (1 x 107) were incubated with mAb 2c/2 for 30 min at 4°C in a total volume of 100 µl. Cells were washed twice in ice-cold PBS/2% BSA and subsequently incubated with anti-mouse-IgG2a/b F(ab')2-coated beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were washed in PBS/2% BSA at 4°C before transfer to MACS columns (Miltenyi Biotec). Columns were washed three times with PBS/2% BSA before cells were flushed out.

Transient transfection of cells

COS-7 cells were transfected with the liposomal transfection reagent DOSPER (Roche, Mannheim, Germany). DNA mixed with DOSPER was incubated for 20 min and added to cells. After 48 h, cells were harvested and subjected to Western blot analysis or metabolic radiolabeling.

Metabolic radiolabeling, immunoprecipitation, SDS-PAGE and Western blotting

For metabolic labeling, 5 x 106 HSV-1-infected cells were starved for 45 min in methionine-free RPMI 1640, followed by a 15-min pulse with 50 µCi [35S]methionine. In some experiments, cells were recultured in medium containing 150 µg/ml nonradioactive methionine for up to 5 h.

For immunoprecipitation, cells were lysed in 0.5% Nonidet P-40 (NP40; Sigma-Aldrich) containing the protease inhibitors aprotinin, PMSF, and trypsin inhibitor (Sigma-Aldrich). Cell debris was removed by centrifugation, and lysates were precleared by precipitation with CL4B-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ). Supernatants were immunoprecipitated with 25 µl of 20-fold-concentrated hybridoma supernatant and protein A-Sepharose. Protein A-bound immunoprecipitates were washed three times with 0.25% NP40 in TBS and subsequently separated by SDS-PAGE and stained for Western blotting.

For immunoblotting, cells were lysed in 0.5% NP40 buffer, electrophoresed, and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membrane was blocked with RotiBlock (Roth, Karlsruhe, Germany) and probed with Ag-specific primary Ab. Detection of primary Ab binding was conducted with HRP-coupled Ab and ECL Western blotting reagent (Amersham Pharmacia Biotech). Immunoisolates were deglycosylated with endoglycosidase H (EndoH) and N-glycosidase F (PGNaseF). Digestion was performed in the buffer recommended by the manufacturer. The reaction mixture was incubated for 16 h at 37°C. Samples were analyzed by SDS-PAGE.

Cell surface biotinylation

JESTHOM cells were infected for 24 h with HSV-1 at a moi = 1 and were subsequently biotinylated using a standard protocol. In brief, 1 x 107 cells were suspended in 1 ml of biotinylation buffer (50 mM boric acid and 150 mM NaCl). Ten microliters of sulfosuccinimidyl-6-biotinamido-6-hexanamidohexanoate (10 mg/ml in H2O; Pierce, Rockford, IL) was added and incubated for 15 min. The reaction was stopped by addition of 20 µl of 100 mM NH4Cl. The samples were washed twice in ice-cold PBS and stored at -70°C.

Immunofluorescence microscopy

COS-7 cells were transfected, seeded onto chamber slides (Nunc, Roskilde, Denmark) and cultured for 36 h. Cells were washed with PBS, fixed in PBS/4% paraformaldehyde, washed, and permeabilized with PBS/0.1% Triton X-100 for 10 min. Subsequently, cells were washed and blocked with PBS/5% BSA for 1 h. After incubation with primary Ab overnight at 4°C, cells were washed, incubated with secondary Ab (goat anti-mouse-Alexa Fluor 488, goat anti-mouse-Alexa Fluor 594, and goat anti-rabbit-Alexa Fluor 488; Molecular Probes, Eugene, OR), diluted 1/400 in PBS/0.2% BSA for 1 h at 37°C, mounted with Mowiol (Sigma-Aldrich), and visualized by fluorescence microscopy (Axiophot; Zeiss, Oberkochen, Germany).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HSV-1 infection impairs expression of the MHCII-associated Ii

To study the molecular impact of the HSV-1 polypeptide gB on the MHCII processing pathway, the DR1 homozygous B lymphoblastoid cell line JESTHOM was infected with HSV-1. Since infection of B cells with HSV-1 is inefficient, infected cells were separated using Ab-bound magnetic beads. After 24 h of virus inoculation, infected cells were sorted with gB-specific mAb. Flow cytometry of surface-expressed HSV-1 proteins on separated cells showed that the vast majority of cells were infected (Fig. 1A, upper panel). MHCII (HLA-DR) surface expression, with MHCI expression included as a control, was monitored with FITC-conjugated mAbs L243 and W6/32 (Fig. 1A, middle and lower panels). Infection with HSV-1 resulted in a moderate decrease in surface expression of MHCII and MHCI molecules. It was reported that HSV-1 infection retains MHCI molecules in the ER because of inhibition of TAP-mediated peptide translocation (3). The small level of decrease of MHCI and MHCII surface expression, that we observed within the short time of HSV-1 infection, could be explained by the slow turnover rate of MHC molecules. For presentation of viral peptides however, the pool of MHC polypeptides delivered from the biosynthesis of the cell is of particular importance.



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  		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{alpha} (mAb 1B5) MHCI H chain (mAb HC10), and DM{alpha} (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 films for 4 h (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.

 
The long half-life of MHC molecules (24) may delay extensive reduction of surface class I and class II polypeptides in infected cells. However, expression of MHCII-associated Ii, which exhibits a half-life of only 2–3 h (25), could be affected by HSV-1 infection. To evaluate the total amount of Ii, DR, and DM, gB-expressing cells were separated at 18, 40, or 64 h after HSV-1 infection. The yield of gB-expressing cells was monitored by flow cytometry of infected cells. After separation, almost all cells stained positive for HSV-1 polypeptides (data not shown). Staining of dead cells with propidium iodide revealed that the lytic activity of HSV-1 increases with infection time at 64 h to ~20% of the cells (data not shown). Cell lysates from infected and uninfected cells were separated by SDS-PAGE and analyzed by Western blotting (Fig. 1B). gB expression increases with infection time, suggesting continued gB synthesis. Staining with mAb Bu-43 against the Ii revealed a dramatic reduction of Ii in infected cells, down to ~14% after 64 h, when compared with uninfected cells. Immunostaining for actin confirmed that equivalent numbers of cells were assayed. The total amount of DR{alpha}, MHCI H chain, and DM{alpha} remains unaffected in infected cells. A significant decrease in Ii expression was detected as early as 18 h after infection. However, Ii expression was not reduced 8 h after infection (Fig. 1C). Thus, the vast majority of Ii vanishes between 8 and 18 h of infection. Since Ii plays an important role in Ag processing (26), the loss of Ii expression may contribute to the decline of MHCII Ag presentation by HSV-1-infected cells. The small amount of Ii detected in infected cells by immunoblotting (Fig. 1B) suggests that Ii is rapidly degraded, perhaps by a viral proteinase. The stability of Ii was investigated by a pulse-chase experiment in infected and uninfected cells (Fig. 1D). Cells were pulse-labeled for 15 min with [35S]methionine and chased for the times indicated. The low rate of Ii synthesis in infected cells (Fig. 1D, upper left panel) was opposed to synthesis in uninfected cells (Fig. 1D, upper right panel). Compared with the half-life of Ii in uninfected cells after 4-h exposure (Fig. 1D, upper right panel), infected cells exposure for 12 h (Fig. 1D, middle panel) showed rapid Ii degradation, but the decay is not accelerated upon HSV-1 infection as can be judged from a densitometric scan (Fig. 1D, lower panel). These results suggest that HSV-1 infection reduces Ii biosynthesis and that physiological degradation leads to low levels of Ii expression. The decline of biosynthesis by HSV-1 infection appears to be nonspecific and also concerns MHCI and MHCII polypeptides (27), which we confirmed by immunoprecipitation of [35S]methionine-labeled MHCII molecules from HSV-1-infected cells (data not shown). To eliminate the activity of residual MHCII molecules derived from biosynthesis, HSV-1 may have evolved a strategy to inhibit loading of viral peptides to newly synthesized MHCII polypeptides to abolish Ag presentation. Therefore, it is possible that HSV-1 in addition targets the MHCII processing pathway.

Peptide loading to MHCII heterodimers is impaired in HSV-1-infected cells

Ag-specific T cell stimulation is sensitive to a low number of MHCII-presented epitopes. The level of biosynthesis in HSV-1-infected cells might permit Ag processing and support presentation by MHCII molecules at levels sufficient to stimulate CD4+ T cells. It was unclear whether generation of peptide MHCII complexes is affected during HSV-1 infection. To evaluate the ability of infected B lymphoblastoid cells to present Ag, the level of SDS-stable MHCII complexes, which represent a fraction of peptide-loaded MHCII heterodimers, was examined in lysates from infected and uninfected cells by immunoblotting (Fig. 2A). Nonboiled samples resolved into SDS-resistant DR complexes and monomeric bands, which stained with DR{alpha}-specific mAb 1B5. SDS-stable DR complexes were strongly reduced in infected cells compared with uninfected cells (Fig. 2A, lanes 1 and 3). In lysates from infected cells, most DR{alpha} appears as monomeric polypeptide, while in uninfected cells, about half of the DR{alpha} is found in SDS-stable complexes. After boiling the samples, which causes dissociation of the SDS-resistant DR complexes, the intensity of DR{alpha} bands in infected and uninfected cells was similar (Fig. 2A, lanes 2 and 4). Western blots for actin were used to confirm that similar amounts of infected and uninfected cell lysates were analyzed (Fig. 2A, lanes 5 and 6). The result in Fig. 2A suggests that a large amount of the DR polypeptide in HSV-1-infected cells is not associated with peptides, at least not as heat-labile DR complexes.



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  		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 for 1 h at room temperature in SDS sample buffer (nb), followed by SDS-PAGE and immunoblotting with mAb 1B5 (DR{alpha}) (lanes 1–4). Cell lysates were also immunoblotted for actin (lanes 5 and 6). The positions of SDS-resistant DR complexes and actin and DR{alpha} 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{alpha} (1B5, left panel) or with mAb against gB (10B7) and mAb against DM{alpha} (5C1, right panel). The positions of gB, DR{alpha}, and DM{alpha} 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{alpha} (mAb 5C1) and against gB (mAb 10B7). Lysates of gB- and DM-transfected cells were separated in lanes 6–8. Lysate from untransfected cells is shown in lane 5. Lysates were immunoblotted using Abs against gB (lane 6), DM{alpha} (lane 7), and DM{beta} (mAb DMK.8) (lane 8). The positions of gB, DM{alpha}, and DM{beta} 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 1–4). The immunoblotted polypeptides were stained with a pool of Abs against Ii (Bu-43) and gB (10B7). Cell lysate was separated in lanes 5–7 and immunoblotted with mAbs against Ii (lane 5), DR{alpha} (lane 6), and DR{beta} (lane 7). The positions of gB, as well as Ig H and L chains are indicated on the left and DR{alpha}, Ii, and DR{beta} on the right.

 
In HSV-1-infected B cells gB associates with DR and with DM heterodimers

HSV-1-infected B cells were lysed with NP40 and immunoprecipitated for gB. Immunoblotting was conducted with Abs against DR{alpha} and against gB. Fig. 2B (left panel) shows that DR was coisolated with gB (lane 1). Expression of gB and DR is demonstrated by immunoblotting of cell lysates (Fig. 2B, left panel, lanes 3 and 4). It appeared possible that gB might also bind to the MHCII-like molecule DM. DM, a peptide binding editor, is required to render the MHCII groove susceptible for peptide binding. To explore a possible interaction between gB and DM under physiological conditions, B lymphoblastoid cells were infected with HSV-1 or were left uninfected, lysed, and immunoprecipitated using mAb against gB (Fig. 2B, right panel). We assumed that the potential binding of gB to DM was similar to the binding of Ii to DM. Therefore, since detection of Ii binding to DM depends on mild detergent, cells were lysed with digitonin. Immunoprecipitates (Fig. 2B, right panel, lanes 1 and 2) and lysates (lanes 3 and 4) were separated by SDS-PAGE followed by Western blotting with Abs directed to gB and DM{alpha}. To exclude that the interaction of DM to gB occurs via DR molecules, we tested binding in the absence of DR. COS-7 cells were transfected with gB and DM, lysed with digitonin, and immunoprecipitated for gB. Again, DM{alpha} was detected by Western blot, confirming association of gB with DM (data not shown). Lysates separated in Fig. 2B, right panel, lanes 3 and 4, show expression of DM and verify expression of gB in infected cells. Lane 1 shows that DM{alpha} was coprecipitated with gB, indicating that gB binds to DM molecules. Binding of gB to DM was not detected after mixing of cell lysates from gB- or DM-transfected COS-7 cells and subsequent immunoprecipitation of DM or gB (Fig. 2C, lanes 2 and 4). This result confirms that the association of gB with DM occurs in infected cells. Immunoblotting of the lysates indicates expression of either gB or DM{alpha}{beta} chains (Fig. 2C, lanes 6–8).

HSV-1-encoded gB competes with Ii for binding to MHCII heterodimers

Recently, we found that the HSV-1 envelope protein gB contains a sequence motif that is also contained in Ii (14). It was possible that Ii and gB compete for binding to MHCII molecules. Alternatively, Ii and gB may bind to the same DR heterodimer. To examine interactions among Ii, gB, and DR, COS-7 cells were transfected with the corresponding cDNAs. Fig. 2D shows immunoprecipitates and cell lysates separated by SDS-PAGE. Immunoblotting of the lysates indicates that Ii, DR{alpha}, and DR{beta} are expressed (Fig. 2D, lanes 5–7). Immunoprecipitates of Ii and gB from transfected and nontransfected COS-7 cells were separated in Fig. 2D, lanes 1–4. Western blotting of the SDS-PAGE separated polypeptides revealed that Ii and gB were independently isolated (Fig. 2D, lanes 2 and 4). Ii and gB do not coisolate through binding to DR. Thus, there are two DR populations, one composed of Ii and DR, and one with DR and gB. However, there are no DR complexes containing both gB and Ii. One can thus conclude that a decrease of Ii and an increase in gB expression, that was observed upon HSV-1 infection, out-competes Ii in newly synthesized DR complexes by gB.

gB migrates with associated DR or DM polypeptides from the ER to Golgi compartments

The fate of gB-associated DR and DM after biosynthesis remains unclear. It has been found that HSV-1 infection retains MHCI molecules in the ER and prevents intracellular transport and presentation of peptides at cell surfaces (28). We examined whether gB-associated DR and DM molecules are exported from the ER. Both DM and DR {alpha}-chains contain two N-linked carbohydrates. In uninfected cells, one of the two glycans of each polypeptide is converted in the Golgi complex from an EndoH-sensitive to an EndoH-resistant form, whereas the second glycan chain remains EndoH sensitive (Fig. 3A, lane 5 and B, lane 4). DR and DM molecules coprecipitated with gB also show resistance of one carbohydrate chain to EndoH treatment (Fig. 3, A and B, lane 2). PNGaseF treatment of cell lysates and immunoprecipitated DR molecules remove both glycan chains from DR{alpha} and DM{alpha} (Fig. 3A, lanes 3 and 6, and B, lane 5). The resistance of one of the glycan chains to EndoH treatment indicates that gB-associated DM and DR polypeptides travel from the ER to Golgi compartments, where the carbohydrates are modified. This is similar to migration of DM and DR molecules in uninfected cells.



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  		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 glycoproteins. 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). Lysates 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 polypeptides was conducted with mAb 1B5 against DR{alpha} (A) or mAb 5C1 against DM{alpha} (B). The positions of DR{alpha}, DM{alpha}, 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 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 incubated with gB mAb 2c/2, which was labeled with Alexa Fluor 594-conjugated anti-mouse Ab, followed by incubation with FITC-conjugated DR Ab Tü-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. Colocalization 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.

 
To assess subcellular localization of DR and DM in gB-expressing cells, COS-7 cells were transfected with cDNAs encoding DR, DM, gB, and as a control, Ii. Cells were seeded on chamber slides, fixed, and permeabilized before staining. Fig. 3C shows immunostaining of DR-, gB-, or Ii-expressing COS-7 cells. The nuclei were counterstained with bis-benzimide. Cells solely expressing DR molecules (Fig. 3I, top) show staining of the perinuclear membrane and polarized distribution at intracellular membranes. This pattern is characteristic of ER labeling as we showed by costaining for the ER marker protein disulfide-isomerase (data not shown). Upon coexpression of DR with Ii, staining for both DR and Ii polypeptides is present in small vesicles distributed uniformly throughout the cytoplasm (Fig. 3I, middle and bottom). gB shows a staining pattern similar to that of DR coexpressed with Ii (Fig. 3II, top). However, when coexpressed with DR, the distribution of gB vesicular staining is strongly reduced and does not extend to the cell membrane (Fig. 3II, bottom). A similar staining was obtained for DR (Fig. 3II, middle). Expression of DM or DM and gB (Fig. 3III) resulted in a subcellular distribution similar to DR/Ii in the first and second panel, except that vesicular staining of DM throughout the cytoplasm did not depend on Ii. Coexpression of gB and DM altered the subcellular distribution of the stained polypeptides (Fig. 3III, middle and bottom). To demonstrate colocalization of DR and gB, cells were stained with FITC anti-DR and anti-gB (red; IV, top and middle). Merging of the red and green stains (IV, bottom) demonstrated colocalization of DR and gB in subcellular vesicles. Expression of DR in gB-expressing cells resulted in a more distinct vesicular label than DR staining in the absence of Ii. This result is consistent with our finding that the carbohydrate of DR{alpha} acquires resistance to EndoH treatment, indicating migration of DR to Golgi compartments.

To demonstrate intracellular localization of gB and MHCII molecules, we used cathepsin B (CatB) as a lysosomal/endosomal marker (Fig. 3D). Coexpression of DR with Ii demonstrates expression of DR in CatB-containing compartments (yellow staining, I, bottom). The colocalization of DR with CatB is strongly reduced in the presence of gB (compare yellow staining of I with II, both bottom). Similar results were obtained when DM and gB were coexpressed. DM exhibits a lysosomal/endosomal distribution, which in contrast to DR is achieved in the absence of Ii (yellow staining, III, bottom). This can be explained by an endosomal sorting signal present on the DM{beta} chain. The endosomal localization of DM is strongly altered when coexpressed with gB (IV, bottom). Some coexpression of DM and CatB is still detected in the presence of gB. However, a high amount of DM is intracellularly localized, different from lysosomal/endosomal staining (red vs yellow staining, IV, bottom).

In addition, gB-, DR-, and Ii-transfected COS-7 cells and HSV-1-infected B lymphoblastoid cells were surface biotinylated. Surface-labeled gB was immunoprecipitated from DR/gB-transfected COS-7 cells (Fig. 3E, lane 3). No surface-labeled DR coprecipitated with gB is visible in lane 3. Expression of DR in gB/DR-transfected cell lysates was confirmed by Western blotting (data not shown). The position of SDS-PAGE-separated gB and DR bands is shown in lanes 1 and 2. Immunoprecipitation of endogenously expressed and surface-biotinylated MHCI (lane 4) did not exhibit association with gB. Precipitation of small amounts of actin (lane 5) revealed that a low quantity of intracellular protein was labeled with biotin. A long exposure of the gel exhibits small amounts of DR bands in lane 3 (data not shown). Consistent with labeling of small amounts of actin, the appearance of these DR bands can be explained by biotinylation of intracellular proteins that were derived from some death cells. The result in Fig. 3E suggests that gB-associated DR molecules are not expressed on the cell surface.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent research indicates that MHCII molecules and the Ag processing pathway are targets for virus evasion strategies. The HIV-encoded Nef protein inhibits cell surface expression of MHCII molecules and presentation of peptides (29). Presumably, Nef affects intracellular trafficking of MHCII polypeptides. The herpes virus human CMV (HCMV) impairs stability of DR and DM molecules and down-modulates DR surface expression during latent infection (30, 31) In mice, it was demonstrated that the HCMV protein US2 binds to MHCII {alpha}{beta} Ii complexes and blocks protein trafficking. US2 promotes degradation of DR{alpha} and DM{beta} chains. In contrast to US2, HSV-1 encoded gB binds only to Ii-free MHCII heterodimers. It was reported, however, that HCMV-derived US3 competes with Ii for binding to MHCII molecules (32). The binding site of US3 to MHCII has not yet been identified. As shown in HSV-1-infected cells, reduction of Ii levels is a way for the virus to limit Ag processing. Similar observations have been made with HIV-2-infected cells. The HIV-2 Vpx polypeptide interacts with Ii, thereby reducing the level of Ii (33).

In a recent study, the reduced capacity of infected B lymphoblastoid cells to stimulate T cells was demonstrated (13). T cells specific for HSV-1-encoded gB and gD polypeptides were used in this study. HSV-1-infected B lymphoblastoid cells used as APCs showed impaired stimulation of the gB- and gD-specific CD4+ T cells. Since only the infected B cells present the Ag the authors provide evidence that HSV-1 inhibits the capacity of lymphoblastoid B cells to activate Ag-specific T cells.

It has been previously reported that HSV-1 infection impairs protein biosynthesis. Soon after infection the HSV-1-derived vhs protein, which possesses RNase activity, induces nonspecific mRNA degradation (34). In addition, viral protein ICP34.5 blocks protein synthesis by dephosphorylating the {alpha} subunit of translation factor 2 (35). Reduced MHC polypeptide synthesis in HSV-1-infected glioblastoma cells was demonstrated (27). Glioblastoma cells show some decrease in MHCII cell surface expression although the total amount of MHCII is constant. This result is consistent with our observation that HSV-1 infection does not alter total MHCII expression in B lymphoid cells. The data of Trgovcich et al. (27) further suggest that MHCII cell surface expression is controlled by an unknown mechanism through the HSV-1 genes UL 41 and {gamma}1 34.5.

HSV-1 is an important human pathogen that infects epithelial cells and sensory ganglia in particular, but also infects other cell types with varying efficiencies. After an acute episode, the virus retracts to the neural ganglia and remains in a latent state. HSV-1 has developed multiple strategies to escape and persist within the host immune system. During primary infection and numerous cycles of reactivation, HSV-1-encoded proteins modify immune responses to virus-infected cells. Virus evasion strategies also affect functional recognition of APCs. HSV-1 infection blocks maturation of dendritic cells by inhibiting the signaling pathway, which results in a reduced capacity to stimulate allogeneic T cells (36). It is interesting to note that in the absence of CD8+ T cells, a vaccine-induced immunity to HSV-1 infection can be achieved (37). In mice, the ability to control HSV-1 infection is attributed to helper T cells. Susceptibility of CD4 T cell-deficient mice to HSV-1 infection challenges the primary importance of this T cell subset and emphasizes the significance of Abs in mediating protection from HSV (38, 39), while the importance of anti-HSV-1 Abs in humans is less clear. Ab-producing B cells might be a potential virus target of HSV-1.

Down-regulation of MHCII and Ii biosynthesis, such as that observed upon HSV-1 infection of B lymphoblastoid cells, shows some similarity to the maturation of Langerhans cells (LC). LC internalize Ags that penetrate skin lesions and subsequently move to lymph nodes, where they present antigenic epitopes to T cells. Under experimental conditions, LC display a differentiation pattern that resembles their in vivo phenotype as professional APCs (40). LC isolated from skin and cultured for several days change their phenotype. Upon tissue extraction of LC, biosynthesis of MHCII and Ii is arrested. The ability to process Ag declines with the decay of Ii. In the absence of Ii, degradation of mouse H2-M is increased (41). Mature LC express MHCII molecules that have acquired their peptides as immature LC. Our results suggest that HSV-1 employs a similar but distinct mechanism to modulate MHCII Ag presentation. In HSV-1-infected cells, the MHCII processing pathway is blocked because Ii vanishes. In addition, gB binds to DM and possibly separates the peptide editor from the MHCII pathway. The small amount of SDS-resistant DR complexes detected in infected cells suggests that peptides loaded onto MHCII heterodimers before infection can still be presented. By affecting the biosynthetic route of DR molecules, however, the aim of the virus is achieved: presentation of viral peptides to CD4+ T cells is impaired.

gB contains a DR1-binding motif at the N terminus, which is of interest, because gB is a target for CD4+ T cells (42). The DR1-binding sequence is flanked by a proline/lysine-rich sequence that is also contained in Ii. The DR1-binding sequence and the proline/lysine-rich sequence of gB resemble the MHCII-binding sequence of Ii (14). Similar to the binding of gB to MHCII, a superantigen from mouse mammary tumor virus imitates the interaction between Ii and MHCII heterodimers (43).

It can be concluded that binding of gB to MHCII heterodimers inhibits peptide loading and prevents presentation of viral peptides. HSV-1-encoded gB is an envelope protein required for infection (44). gB attaches to negatively charged heparan sulfate moieties and promotes fusion of the viral envelope with the cell membrane, followed by entry of virions into cells. The finding within this study that DR expression changes the intracellular distribution of gB may have consequences for virus particle production. gB polypeptides engaged in DR binding may not be available to serve as virus envelope proteins. Therefore, DR expression could reduce HSV-1 replication in APCs.

The 874-aa sequences of gB from various HSV-1 strains show high homology with a few patches of polymorphism. Two gB polymorphic residues are located in the PKPPKP sequence, the site where gB binds to MHCII heterodimers. These sequence variations can be related to a mouse infection model. It has been reported that infection with the HSV-1 strain ANGpath is lethal to mice, whereas strain HSZP is nonpathogenic (45, 46). Through transfer and mapping studies, this pathogenic phenotype could be attributed to the gB molecule (45, 47). The gB sequence of ANGpath is highly similar to HSV-1 strain 17, which was used in this study. In contrast, HSZP and other nonpathogenic HSV-1 strains show four mutations at the N terminus of gB between aa residues 59 and 79 (48). Two residues, Pro77 and Lys79, of ANGpath were mutated in the nonpathogenic strains. These residues are also located in the MHCII binding site PKP77PK79P of gB from strain 17 (49). The binding properties of gB to MHCII molecules could be related to the pathogenic phenotype of HSV-1 strain ANGpath. Moreover, infection of mice with HSV-1 strains KOS or F results in a latent infection only with strain F, whereas spreading of the KOS strain is not controlled (50). The MHCII biosynthetic pathway in HSV-1-infected cells appears to be interrupted in cells infected with the KOS strain. As shown in this report, gB efficiently interacts with MHCII molecules in HSV-1-infected cells. A mutation of Pro80 in gB (strain F) to Thr80 (strain KOS), which is present in a MHCII-binding sequence, could account for the variant interaction to mouse Ia molecules in cells infected with strains KOS and F.

HSV-1 infection is endemic within the human population. Many individuals carry the virus without any apparent disease. In contrast, others suffer from frequent virus reactivation and recurrent disease. One may assume that susceptibility to HSV-1 reactivation is linked to a partial deficient immunity. It is tempting to speculate that HLA polymorphism is associated with immunity to HSV-1 infection. The molecular basis for HSV-1-related immunity could involve interaction of the virus envelope protein gB with polymorphic MHCII molecules. In early studies, natural resistance to herpes virus infection was linked to MHC haplotypes by examining H2-congenic mouse strains (51). There is evidence, that the human T cell response to gB is influenced by MHC, but only limited experimental data on HLA typing and HSV-1 infection are available (52, 53). Investigation of the genetic basis of HSV-1 immunity might be an interesting topic for future studies.


   Acknowledgments
 
We thank Dr. G. Moldenhauer for providing Abs.


   Footnotes
 
1 This work was supported by a Grant Ko 810/6-1 from the Deutsche Forschungsgemeinschaft. Back

2 Address correspondence and reprint requests to Dr. Norbert Koch, Universität Bonn, Abteilung Immunbiologie, Institut für Molekulare Physiologie Römerstrasse 164, 53117 Bonn, Germany. E-mail address: norbert.koch{at}uni-bonn.de Back

3 Abbreviations used in this paper: MHCI and II, MHC class I and class II; HSV-1, HSV type 1; Ii, invariant chain; ER, endoplasmic reticulum; gB, glycoprotein B; NP40 NonidetP-40; EndoH, endoglycosidase H; PGNaseF, N-glycosidase F; CatB, cathepsin B; moi, multiplicity of infection; PVDF, polyvinylidene difluoride; HCMV, human CMV; LC, Langerhans cell. Back

Received for publication April 17, 2003. Accepted for publication July 14, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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