Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Oct 14;90(1):356-67.
doi: 10.1128/JVI.02183-15. Print 2016 Jan 1.

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II

Laura L Quinn  1 , Luke R Williams  1 , Claire White  1 , Calum Forrest  1 , Jianmin Zuo  2 , Martin Rowe  1
Affiliations

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II

Laura L Quinn et al. J Virol. .

Abstract

The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8(+) cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8(+) cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8(+) cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4(+) cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8(+) and CD4(+) T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner.

Importance: Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8(+) T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8(+) T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8(+) T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4(+) T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Screening of EBV lytic genes to identify potential MHC class I immune evasion genes. MJS cells were transiently transfected with pCDNA3.1-IRES-GFP plasmids carrying a selection of EBV lytic genes. At 24 h posttransfection, surface levels of MHC class I (A) and MHC class II (B) on GFP-positive cells were analyzed using two-color flow cytometry. (C) AKBM cells were induced into the lytic cycle by cross-linking of B cell receptors for 1 h at 37°C and were analyzed by Western blotting at the indicated time points postinduction. Levels of BZLF1 (upper blot), BDLF3 (middle blot; stars indicate monomeric and trimeric forms of BDLF3), and the loading control calregulin (lower blot) are shown.
FIG 2
FIG 2
BDLF3 expression induces the downregulation of surface MHC class I and MHC class II. MJS cells (A to D) and DG75 cells (E to H) were transiently transfected with the control-GFP or BDLF3-GFP plasmid. At 24 h posttransfection, two-color flow cytometry was used to measure surface levels of MHC class I (A and E), MHC class II (B and F), TfR (C and G), ICAM1 (D), and CD19 (H) in the GFP+ populations of control-GFP-transfected cells (solid lines) and BDLF3-GFP-transfected cells (dashed lines). The gray histograms denote background staining obtained with an isotype control antibody.
FIG 3
FIG 3
BDLF3 induces downregulation of all HLA class I and class II alleles. (A) The MHC class I-negative cell line K562, transduced to stably express either HLA-A2, -B35, or -Cw1, was electroporated with the control-GFP or BDLF3-GFP plasmid. At 24 h posttransfection, two-color flow cytometry was used to measure surface MHC class I levels in the GFP+ populations in the control-GFP-transfected cells (solid lines) and BDLF3-GFP-transfected cells (dashed lines). (B) HEK-293 cells stably expressing CIITA were transiently transfected with the control-GFP or BDLF3-GFP plasmid. At 24 h posttransfection, two-color flow cytometry was used to measure surface HLA-DR and HLA-DQ levels in GFP+ populations in the control-GFP-transfected cells (solid lines) and the BDLF3-GFP-transfected cells (dashed lines). The gray histograms denote background staining obtained with an isotype control antibody.
FIG 4
FIG 4
BDLF3 can inhibit EBV-specific CD8+ and CD4+ T cell recognition. MJS cells were cotransfected with the p509 plasmid (BZLF1 expression vector) together with control-GFP or BDLF3-GFP. At 24 h posttransfection, the MJS cells were cocultured with effector T cells, i.e., a BZLF1 (RAK)-specific CD8+ T cell clone, for a further 18 h, and the supernatants were tested for the release of IFN-γ as a measure of T cell recognition. All results are expressed as amounts of IFN-γ release (pg/ml), and error bars indicate standard deviations for triplicate cultures. (B) Total cell lysates were generated from the above transfections and analyzed by Western blotting using antibodies specific for BDLF3, BZLF1, and calregulin (loading control). The asterisks adjacent to the BDLF3 blot indicate monomeric and trimeric forms of BDLF3. (C) MJS-DR51 cells were first transfected with EBNA1ΔNLS, allowed to recover in culture overnight, and then divided into two groups and transfected with either BDLF3-NGFR or control-NGFR. After another 24 h, NGFR+ BDLF3+ or control NGFR+ cells were sorted with magnetic beads and used as targets for HLA-DR51-restricted, EBNA1 (SNP)-specific CD4+ T cell clones. Recognition was measured as the amount of IFN-γ release (pg/ml) by T cell clones. Error bars represent standard deviations of the means for triplicate assay replicates. Results are representative of three independent experiments. (D) Total cell lysates were generated from the above transfections and analyzed by Western blotting using antibodies specific for BDLF3, EBNA1, and calregulin (loading control).
FIG 5
FIG 5
BDLF3 induces a more dramatic reduction in surface MHC class I and II than in whole-cell MHC levels. (A) MJS cells were transiently transfected with the control-GFP or BDLF3-GFP plasmid. At 24 h posttransfection, two-color flow cytometry was used to measure the levels of surface MHC class I (upper left), MHC class II (middle left), and ICAM1 (lower left) in the viable GFP+ populations of control-GFP-transfected cells (solid lines) and BDLF3-GFP transfected cells (dashed lines). The gray histograms denote background staining obtained with an isotype control antibody. In parallel, these GFP+ transfected MJS cells were analyzed for whole-cell levels of MHC class I (upper right), MHC class II (middle right), and ICAM1 (lower right), using intracellular staining of fixed and permeabilized cells. The results are representative of repeated experiments. (B) Relative mean fluorescence intensities (MFI) of MHC class I, MHC class II, and ICAM1 were calculated for BDLF3-GFP+ cells compared to control GFP+ cells. Results are combined data from three independent experiments. White bars represent surface staining, and gray bars represent whole-cell staining. Differences that reached significance (P < 0.05) by Student's paired t test are denoted by asterisks.
FIG 6
FIG 6
BDLF3 induces more rapid internalization and a delayed appearance of both MHC class I and class II molecules at the cell surface. Internalization and appearance assays were performed on MJS cells transiently expressing control-GFP or BDLF3-GFP. The GFP+ population was used to gate BDLF3-expressing cells. Internalization and appearance assays were performed on cells pretreated on ice with saturating amounts of anti-MHC class I antibody or anti-MHC class II antibody. Cells were then washed and incubated at 37°C for up to 60 min. (A) For the internalization assay, viable cells harvested at each time point were stained with an APC-conjugated goat anti-mouse IgG antibody and analyzed using flow cytometry at the indicated times; this identified the prelabeled antibody-bound MHC molecules that remained at the surface, while endocytosed labeled MHC molecules were not detected on the viable cells. The mean fluorescence intensities of staining were averaged for triplicate samples and then normalized to the values for time zero samples. (B) For the appearance assays, newly arrived MHC-I and MHC-II molecules, which were not prelabeled with unconjugated antibodies, were detected by staining with an APC-conjugated anti-MHC class I antibody or anti-MHC class II antibody. The mean fluorescence intensities of staining were averaged for triplicate samples and then normalized to the values for time zero samples. Results are representative of three independent experiments.
FIG 7
FIG 7
Treatment of BDLF3-expressing cells with a proteasome inhibitor prevents downregulation of MHC class I and class II. MJS cells were transiently transfected with the BDLF3-GFP or control-GFP plasmid and then incubated in normal medium (A) or MG132 (5 μM)-supplemented medium (B). At 24 h posttransfection, two-color flow cytometry was used to measure surface MHC class I (upper histograms), surface MHC class II (middle histograms), and surface ICAM1 (lower histograms) in GFP+ populations of control-GFP-transfected cells (solid lines) and BDLF3-GFP-transfected cells (dashed lines). The gray histograms denote background staining obtained with an isotype control antibody. Results are representative of repeat experiments (n > 4). (C and D) MJS cells were transiently transfected with the BDLF3-GFP or control-GFP plasmid and then were incubated with MG132 (5 μM). Following drug treatment, the rates of internalization (C) of MHC-I (top panel) and MHC-II (bottom panel) and the rates of appearance (D) of MHC-I (top panel) and MHC-II (bottom panel) were measured using the same methods as those described in the legend to Fig. 6. (E and F) MJS cells were transfected with a ubiquitin expression plasmid plus either the control-NGFR or BDLF3-NGFR plasmid. The transfected cells were then divided into two groups and incubated in normal medium or medium supplemented with MG132. At 24 h posttransfection, NGFR+ BDLF3+ or control NGFR+ cells were sorted with magnetic beads, and surface MHC class I (C) or MHC class II (D) molecules were immunoprecipitated, eluted, and then immunoblotted using an anti-ubiquitin (Ub) antibody (P4D1).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Rickinson AB, Kieff E. 2007. Epstein-Barr virus, p 2655–2700. In Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE (ed), Fields virology, 5th ed Lippincott Williams & Wilkins, Philadelphia, PA.
    1. Ressing ME, Horst D, Griffin BD, Tellam J, Zuo J, Khanna R, Rowe M, Wiertz EJ. 2008. Epstein-Barr virus evasion of CD8(+) and CD4(+) T cell immunity via concerted actions of multiple gene products. Semin Cancer Biol 18:397–408. doi:10.1016/j.semcancer.2008.10.008. - DOI - PubMed
    1. Rowe M, Zuo J. 2010. Immune responses to Epstein-Barr virus: molecular interactions in the virus evasion of CD8+ T cell immunity. Microbes Infect 12:173–181. doi:10.1016/j.micinf.2009.12.001. - DOI - PMC - PubMed
    1. Hislop AD, Ressing ME, van Leeuwen D, Pudney VA, Horst D, Koppers-Lalic D, Croft NP, Neefjes JJ, Rickinson AB, Wiertz EJ. 2007. A CD8+ T cell immune evasion protein specific to Epstein-Barr virus and its close relatives in Old World primates. J Exp Med 204:1863–1873. doi:10.1084/jem.20070256. - DOI - PMC - PubMed
    1. Rowe M, Glaunsinger B, van Leeuwen D, Zuo J, Sweetman D, Ganem D, Middeldorp J, Wiertz EJ, Ressing ME. 2007. Host shutoff during productive Epstein-Barr virus infection is mediated by BGLF5 and may contribute to immune evasion. Proc Natl Acad Sci U S A 104:3366–3371. doi:10.1073/pnas.0611128104. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances