The lytic cycle of Epstein-Barr virus is associated with decreased expression of cell surface major histocompatibility complex class I and class II molecules

Abstract

Human herpesviruses utilize an impressive range of strategies to evade the immune system during their lytic replicative cycle, including reducing the expression of cell surface major histocompatibility complex (MHC) and immunostimulatory molecules required for recognition and lysis by virus-specific cytotoxic T cells. Study of possible immune evasion strategies by Epstein-Barr virus (EBV) in lytically infected cells has been hampered by the lack of an appropriate permissive culture model. Using two-color immunofluorescence staining of cell surface antigens and EBV-encoded lytic cycle antigens, we examined EBV-transformed B-cell lines in which a small subpopulation of cells had spontaneously entered the lytic cycle. Cells in the lytic cycle showed a four- to fivefold decrease in cell surface expression of MHC class I molecules relative to that in latently infected cells. Expression of MHC class II molecules, CD40, and CD54 was reduced by 40 to 50% on cells in the lytic cycle, while no decrease was observed in cell surface expression of CD19, CD80, and CD86. Downregulation of MHC class I expression was found to be an early-lytic-cycle event, since it was observed when progress through late lytic cycle was blocked by treatment with acyclovir. The immediate-early transactivator of the EBV lytic cycle, BZLF1, did not directly affect expression of MHC class I molecules. However, BZLF1 completely inhibited the upregulation of MHC class I expression mediated by the EBV cell-transforming protein, LMP1. This novel function of BZLF1 elucidates the paradox of how MHC class I expression can be downregulated when LMP1, which upregulates MHC class I expression in latent infection, remains expressed in the lytic cycle.

FIG. 1.

Downregulation of MHC class I expression in EBV lytic infection. (A) Surface expression of MHC class I molecules on the latency III and lytic EBV-positive BL cell line Ag876. Viable cells were stained with antibody W6/32 (IgG2a isotype, anti-HLA-A, -B, and-C) and then fixed and permeabilized to allow staining with antibody BZ.1 (IgG1 isotype, anti-BZLF1 immediate-early EBV antigen), followed by detection with a pool of FITC-conjugated anti-IgG1 and RPE-conjugated anti-IgG2a secondary antibodies. The left-hand panel is a dot plot of the flow cytometry results of RPE staining (surface MHC class I molecules) (y axis) and FITC staining (nuclear BZLF1) (x axis). The right-hand panel is a histogram of the RPE staining (surface MHC class I molecules) of the latent, BZLF1⁻ population (light shading) and the lytic, BZLF1⁺ population (dark shading). The MFI of surface MHC class I expression on the latent, BZLF1⁻ cells is 24, whereas the corresponding MFI for the lytic, BZLF1⁺ cell population is 5. Results shown are representative of four independent experiments. (B) Surface expression of MHC class I molecules on the normal EBV-transformed LCL Eli-LCL. Immunofluorescence staining and analysis were performed exactly as for the Ag876 cells in panel A. In this representative experiment, the MFI of surface MHC class I expression obtained for the latent, BZLF1⁻ cells was 28, and the corresponding MFI for the lytic, BZLF1⁺ cells was 8.

FIG. 2.

Analysis of cell surface protein expression in the lytic cycle. Ag876 cells were stained with antibodies specific for a range of cell surface antigens (for MHC class II, MAb WR18; for CD19, MAb BU.12; for CD40, MAb LOB7/6; for CD54, MAb 15.2; for CD80, MAb L307.4; and for CD86, MAb FUN-1) and labeled with RPE, together with MAb BZ.1 against the BZLF1 immediate-early EBV antigen, labeled with FITC. A representative flow cytometry analysis is shown for each combination of antibodies. Dark shading represents cell surface antigen staining on lytic, BZLF1⁺ cells; light shading represents cell surface antigen staining on latent, BZLF1⁻ cells; curves defined by dashed lines represent the background RPE fluorescence obtained on cells stained with control antibodies.

FIG. 3.

Summary of the relative levels of cell surface antigen expression on lytic, BZLF1⁺ cells in Ag876 BL cultures. Each result represents the mean relative intensity from at least three separate experiments and is shown as a percentage of the MFI of RPE staining of latently infected cells in the same experiment. Error bars represent the standard errors of the means from at least three separate experiments.

FIG. 4.

Downregulation of MHC class I expression is an early-lytic-cycle event. (A) Surface expression of MHC class I molecules on BZLF1⁺ and VCA⁺ cells in the lytic cycle. Ag876 BL cells were simultaneously stained to detect surface MHC class I expression and intracellular lytic-cycle antigen expression exactly as for Fig. 1. The left-hand flow cytometry dot plot shows staining with MAbs W6/32 (anti-MHC class I; RPE conjugated) (y axis) and BZ.1 (anti-BZLF1; FITC conjugated) (x axis). The right-hand dot plot shows parallel staining of cells with MAb W6/32 (y axis) and L2 (anti-VCA; FITC conjugated) (x axis). (B) Surface expression of MHC class I molecules on acyclovir-treated and untreated Ag876 BL cells. Cells cultured in acyclovir for 2 weeks and replicate untreated cells were simultaneously stained with MAbs W6/32 and BZ.1 exactly as for Fig. 1. Results shown are dot plot flow cytometry profiles of W6/32 staining (RPE conjugated) (y axis) versus BZ.1 staining (FITC conjugated) (x axis) obtained for untreated control cells (left panel) and acyclovir-treated cells (right panel).

FIG. 5.

LMP1 expression during the lytic cycle. Western blot analysis of LMP1 expression in EBV lytic cycle-positive P3HR1-c16 cells was performed. Cells were cotransfected with 8 μg of a BZLF1 expression plasmid to induce the EBV lytic cycle and with 3 μg of a rat CD2 surface antigen expression plasmid to allow transfected cells to be positively selected by immunomagnetic separation. At 24 h following transfection, viable cells were labeled with OX34 (an anti-rat CD2 MAb) followed by FITC-conjugated anti-IgG; then they were immunomagnetically sorted with anti-FITC MACS microbeads. BZLF1-transfected cells were positively sorted on the basis of rat CD2 expression, while the unbound flowthrough cells were taken to be untransfected, latent cells. Cell samples were analyzed by Western immunoblotting. When probed with EE human serum (top panel), the blot showed staining of BZLF1, early antigens, and late antigens in the positively sorted cells and not in the untransfected, flowthrough cells. Reprobing the blot for LMP1 with MAbs CS.1-4 (lower panel) demonstrated induced expression of LMP1 in the positively sorted, lytically infected cells.

FIG. 6.

BZLF1 inhibits LMP1-mediated upregulation of MHC class I expression. (A) Flow cytometric analysis of surface MHC class I expression in LMP1-transfected EBV-negative DG75 cells. Each transfection included 1 μg of the GFP expression vector pEGFP-N1, cotransfected with either 4 μg of pSG5 empty vector DNA, 4 μg of an LMP1 expression plasmid, 4 μg each of an LMP1 expression plasmid and a BZLF1 expression plasmid, or 4 μg each of an LMP1 expression plasmid and an IκBαΔN expression plasmid. The total amount of DNA per transfection was kept constant by addition of an appropriate amount of pSG5 empty vector DNA. MHC class I expression was detected 48 h following transfection by staining cells with MAb Tü149 followed by RPE-conjugated anti-IgG antibodies and analyzing by flow cytometry. The MFI of RPE staining (cell surface MHC class I molecules) on the GFP-positive (transfected) viable cell population was quantified. Results shown are means and standard errors from triplicate experiments. (B) Effect of BZLF1 and IκBαΔN on constitutive surface MHC class I expression in EBV-negative cells. DG75 cells were cotransfected with 1 μg of a marker plasmid (the GFP expression vector pEGFP-N1) together with 4 μg of either pSG5 empty vector DNA, a BZLF1 expression plasmid, or an IκBαΔN expression plasmid. At 48 h following transfection, the cells were stained for MHC class I expression exactly as for Fig. 6A, and the MFI of RPE staining (cell surface MHC class I molecules) on the GFP-positive (transfected) viable cell population was quantified. Results shown are means and standard errors from triplicate experiments.

FIG. 7.

Effect of BZLF1 on LMP1-mediated activation of NF-κB. (A) Dose-dependent inhibition of NF-κB activation by BZLF1. As much as 6 μg of a BZLF1 expression plasmid was cotransfected with 3 μg of the NF-κB reporter 3Enh-Luc in DG75 cells. The total amount of DNA per transfection was kept to a constant 9 μg by addition of an appropriate amount of empty vector DNA. Luciferase activity was measured after 24 h. Results shown are means of results from duplicate experiments. (B) Effect of BZLF1 on LMP1-mediated NF-κB activation. DG75 cells were cotransfected with 3 μg of the NF-κB reporter 3Enh-Luc together with either 4 μg of pSG5 empty vector DNA, 4 μg of an LMP1 expression plasmid, 4 μg each of an LMP1 expression plasmid and a BZLF1 expression plasmid, or 4 μg each of an LMP1 expression plasmid and an IκBαΔN expression plasmid, exactly as for Fig. 6A. The total amount of DNA per transfection was kept constant by addition of an appropriate amount of empty vector DNA. Luciferase activity was measured after 24 h. Results shown are means and standard errors from triplicate experiments.