The Epstein-Barr virus G-protein-coupled receptor contributes to immune evasion by targeting MHC class I molecules for degradation - PubMed (original) (raw)

The Epstein-Barr virus G-protein-coupled receptor contributes to immune evasion by targeting MHC class I molecules for degradation

Jianmin Zuo et al. PLoS Pathog. 2009 Jan.

Abstract

Epstein-Barr virus (EBV) is a human herpesvirus that persists as a largely subclinical infection in the vast majority of adults worldwide. Recent evidence indicates that an important component of the persistence strategy involves active interference with the MHC class I antigen processing pathway during the lytic replication cycle. We have now identified a novel role for the lytic cycle gene, BILF1, which encodes a glycoprotein with the properties of a constitutive signaling G-protein-coupled receptor (GPCR). BILF1 reduced the levels of MHC class I at the cell surface and inhibited CD8(+) T cell recognition of endogenous target antigens. The underlying mechanism involves physical association of BILF1 with MHC class I molecules, an increased turnover from the cell surface, and enhanced degradation via lysosomal proteases. The BILF1 protein of the closely related CeHV15 gamma(1)-herpesvirus of the Rhesus Old World primate (80% amino acid sequence identity) downregulated surface MHC class I similarly to EBV BILF1. Amongst the human herpesviruses, the GPCR encoded by the ORF74 of the KSHV gamma(2)-herpesvirus is most closely related to EBV BILF1 (15% amino acid sequence identity) but did not affect levels of surface MHC class I. An engineered mutant of BILF1 that was unable to activate G protein signaling pathways retained the ability to downregulate MHC class I, indicating that the immune-modulating and GPCR-signaling properties are two distinct functions of BILF1. These findings extend our understanding of the normal biology of an important human pathogen. The discovery of a third EBV lytic cycle gene that cooperates to interfere with MHC class I antigen processing underscores the importance of the need for EBV to be able to evade CD8(+) T cell responses during the lytic replication cycle, at a time when such a large number of potential viral targets are expressed.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. BILF1 identified as a lytic gene that downregulates surface MHC class I.

293 (A) or MJS (B) cells were transfected with different EBV genes in the bi-cistronic vector, pCDNA3-IRES-nlsGFP. At 48 hr post-transfection, surface MHC class I was stained with PE-conjugated W6/32 mAb and (in MJS only) MHC class II was stained with PE-conjugated anti-DR mAb, YE2/36-HLK. Two-colour flow cytometry was used to analyse staining in the untransfected GFP− population, shown as the solid line histogram, and in the transfected GFP+ population, shown as the dashed line histogram. The grey histogram denotes background staining obtained with an isotype control PE-conjugated antibody.

Figure 2

Figure 2. Characterization of cells stably transduced with a BILF1 retroviral vector.

(A) 293 or MJS cells were stably transduced with control (pQCXIH) or BILF1 (pQCXIH-HABILF1) retrovirus. Surface MHC class I molecules were stained with PE-conjugated W6/32 antibodies and analyzed by flow cytometry. The solid line histograms depict the surface HLA class I staining of control cell lines, while the dashed line histogram depicts the surface HLA class I staining of cell lines expressing BILF1. The grey histogram illustrates background staining obtained with an isotype control PE-conjugated antibody. (B) Total cell lysates were generated from the retrovirus-transduced 293 and MJS cell lines, and 2×105 cell equivalents were separated by SDS-PAGE and analyzed by Western Blotting with mAbs specific for BILF1 (3F10, anti-HA tag), MHC class I (HC10), MHC class II (DA6.147), TAP-1 (148.3), TAP-2 (435.3), TfR (H68.4) or with polyclonal antibodies to calregulin as a loading control.

Figure 3

Figure 3. BILF1 inhibits T cell recognition of endogenous EBV antigen in MJS cells.

(A) MJS cells were co-transfected with 0.02 µg p509 plasmid (BZLF1 expression vector) and different amounts (0–2 µg) of pCDNA3-HABILF1-IRES-nlsGFP bulked to a constant amount of DNA with control plasmid. At 24 hr post-transfection, the MJS cells were co-cultured with CD8+ effector ‘RAK’ T cells for a further 18 hrs and the supernatants were tested for the release of IFN-γ as a measure of T cell recognition. All results are expressed as IFN-γ release in pg/ml and error bars indicate standard deviation of triplicate cultures. (B) Total cell lysates were generated from the above transfections, and 2×105 cell equivalents were separated and analyzed by Western Blotting using antibodies specific for BZLF1, HA tag (BILF1), or calregulin as a loading control.

Figure 4

Figure 4. BILF1 downregulates surface MHC class I expression and inhibits the T cell recognition of endogenous EBV antigen in LCLs.

(A) LCLs were transduced with PLZRS-HABILF1-IRES-GFP retrovirus. After 6 days, surface MHC class I was stained with PE-conjugated W6/32 mAb and MHC class II was stained with PE-conjugated anti-DR mAb, YE2/36-HLK. Two-colour flow cytometry was used to analyze staining in the untransduced, GFP−, population, shown as the solid line histogram, and in the transduced GFP+ (BILF1+) population, shown as the dashed line histogram. The grey histogram denotes background staining obtained with an isotype control PE-conjugated antibody. (B) LCL cultures transduced with control retrovirus or with the BILF1 retrovirus were sorted by flow cytometry to generate GFP+/BILF1− and GFP+/BILF1+ lines to use as targets in assays with EBV-specific T cells. The control and BILF1+ LCL targets were incubated with HLA-matched CD8+ effector T cells clones specific for EBNA1 (HPV), EBNA3A (YPL), or LMP2A (CLG) peptides, or a CD4+ effector T cell clone specific for a BHRF1 (PYY) peptide. After 18 hrs the supernatants were tested for the release of IFN-γ as a measure of T cell recognition. All results are expressed as IFN-γ release in pg/ml and error bars indicate standard deviation of triplicate cultures.

Figure 5

Figure 5. Effect of BILF1 on maturation and degradation of MHC class I molecules.

(A) Acquisition of endoglycosidase H (Endo H) -resistance of MHC class I heavy chain. 293 cells (2×106) stably transduced with control or BILF1 retrovirus were metabolically labeled for 15 min with 35S-methionine/cysteine and chased for the indicated time periods. After lysis in NP-40 detergent buffer, samples were immunoprecipitated with mAb W6/32 and treated with Endo H enzyme. Protein samples were separated by 10% acrylamide SDS/PAGE gel, dried and exposed to autoradiography. (B) Kinetics of MHC class I molecule degradation. 293 cells (2×106) stably transduced with control or BILF1 retrovirus were metabolically labeled for 15 min and chased for the indicated time periods. After lysis in NP-40 detergent buffer, samples were immunoprecipitated with mAb W6/32 (HLA class I heavy chain and β2-microglobulin) or H68.4 (TfR). Protein samples were separated by 10% SDS/PAGE gel, dried and exposed to autoradiography.

Figure 6

Figure 6. BILF1 is physically associated with the MHC class molecule complex.

(A) 293 cells (107) stably transduced with control (

c

) or BILF1 (

b

) retrovirus were metabolically labeled for 15 min and chased for 20 min. After lysis in NP-40 buffer and immunoprecipitation with mAb W6/32, the samples were dissociated by boiling in reducing sample buffer, and were re-precipitated with either 3F10 ( HA tag on BILF1) or HC10 (MHC class I heavy chain). Protein samples were separated by 10% acrylamide SDS/PAGE gel, dried and exposed to autoradiography. The arrowhead and bracket indicate the presence of 33 kD and 45–55 kD BILF1 bands, whilst the asterisks indicate probable ubiquitinated MHC class I species. (B) 293 cells (2×106) stably transduced with control (

c

) or BILF1(

b

) retrovirus were treated with concanamycin A (50 nM) for 20 hr prior to lysing the cells with NP40 detergent buffer and immunoprecipitation with antibodies specific for MHC class I (W6/32), HA tagged BILF1 (12CA5), or TfR (H68.4). Cell lysates and immune complexes were separated by SDS/PAGE gel, and analyzed by western blotting using antibodies specific for MHC class I (HC10), HA tagged BILF1 (3F10), and TfR (H68.4). The first two samples on the gel are total cell lysates representing 5% of the input lysate for immunoprecipitations.

Figure 7

Figure 7. BILF1 associates with MHC class I molecules at the cell surface and increases their rate of internalization.

(A) BILF1 is predominantly localized at the cell surface. 293 cells stably transduced with BILF1 retrovirus were grown on glass slides, fixed and permeabilized, then stained with rat anti-HA (3F10) primary antibodies and Alexa Fluor® 594 goat anti-rat IgG. The nuclei were counterstained with DAPI. The stained slides were analyzed with a laser scanning confocal microscope, and the three photographs show different 1 micron-thick sections through representative cells. BILF1 stained red, and the nuclei stained blue. (B) BILF1 and MHC class I molecules co-precipitate at the cell surface. 293 cells (2×106) stably transduced with control (

c

) or BILF1 (

b

) retrovirus were incubated with saturating concentrations of antibodies specific for MHC class I (W6/32), TfR (H68.4) or HA tagged BILF1 (3F10) on ice. After washing away excess antibody, the cells were lysed with NP40 detergent buffer, then precipitated with protein A/G beads and subjected to western-blotting as in Fig. 6B, using antibodies specific for MHC class I (HC10) and HA tagged BILF1 (3F10). (C) BILF1 increases the rate of internalization of MHC class I, but not class II, from the cell surface. MJS cells stably transduced with control or BILF1 retrovirus were incubated at 0°C with saturating concentrations of mAb to MHC class I (W6/32; top graph) or MHC class II (L234; bottom graph), then washed and incubated at 37°C for different periods of time. The cells were subsequently stained with PE-conjugated goat anti-mouse IgG antibody, and analyzed by flow cytometry. The mean fluorescence intensities of staining were averaged for triplicate samples, and normalized to the initial time 0 min samples. (D) BILF1 increases the rate of internalization, but not the rate of appearance, of MHC class I at the cell surface. Top graph: 293 cells stably transduced with control or BILF1 retrovirus were incubated at 0°C with saturating concentrations of mAb to MHC class I (W6/32), then treated exactly as for the internalization assay performed with MJS cells in panel C. Bottom graph: replicate aliquots of the saturated W6/32-bound cells were harvested at the indicated time points, and the appearance of new MHC class I molecules was assayed by staining with PE-conjugated W6/32 antibody. The mean fluorescence intensities of staining were averaged for triplicate samples.

Figure 8

Figure 8. Lysosomal inhibitors block BILF1-enhanced degradation of MHC class I.

(A) 293 cells stably transduced with control- (

c

) or BILF1- (

b

) retrovirus were treated with or without Bafilomycin A1, concanamycin A, or leupeptin for 20 hr. Lysates from 2×105 cell equivalents were separated by SDS/PAGE gel, and analyzed by western blotting using antibodies specific for MHC class I (HC10) and calregulin. The blot is one representative of three independent experiments. The histogram shows the mean results (±S.D.) of quantification by densitometry of all the blots from 3 independent experiments, where the densities of the HC10 bands were normalized relative to their own calregulin loading control. (B) 293 cells stably transduced with control or BILF1 retrovirus were treated with concanamycin A (50 nM) for 6 hr prior to fixation and permeabilization with methanol/acetone, then stained with W6/32 primary antibodies and Alexa Fluor® 488 goat anti-mouse IgG secondary antibodies. The photographs were obtained with a conventional fluorescence microscope. (C) 293 cells stably transduced with control (

c

) or BILF1 (

b

) retrovirus were treated with or without the proteasome inhibitor, MG132, for 20 hr, and analyzed by western blot as in panel A. The additional, lower molecular weight species detected is probably deglycosylated and/or partially degraded free heavy chain that is normally targeted for proteasomal degradation.

Figure 9

Figure 9. The ability of BILF1 homologs to downregulate MHC class I.

(A) Multiple sequence alignment of EBV-BILF1, the rhesus lymphocryptovirus CeHV15-BILF1, and KSHV-ORF74. The alignment was done with ClustalW version 1.8, and shading was done with Boxshade version 3.21, available at

http://www.ch.embnet.org/software/BOX\_form.html

. (B) 293 or MJS cells were transfected with EBV-BILF1, CeHV15-BILF1 or KSHV-ORF74 genes in the bicistronic vector, pCDNA3-IRES-nlsGFP. At 48 hr post-transfection, surface MHC class I molecules were stained and analyzed exactly as in Fig. 1.

Figure 10

Figure 10. BILF1 signaling function is not required to downregulate MHC class I.

(A) Wild-type or K122A-mutant BILF1 expression plasmids were transfected into the HEK293-NFκB reporter cell line, and the degree of NFκB activation measured by detection of luciferase activity. The results are the mean±S.D. for three independent experiments performed in triplicate. (B) 293 and MJS cells were transfected with wild type BILF1 or mutant K122A-BILF1 genes in the bicistronic vector, pCDNA3-IRES-nlsGFP. At 48 hr post-transfection, surface MHC class I molecules were stained and analyzed exactly as in Fig. 1.

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