RelB nuclear translocation mediated by C-terminal activator regions of Epstein-Barr virus-encoded latent membrane protein 1 and its effect on antigen-presenting function in B cells

Abstract

Previous studies have shown that Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is uniquely able to up-regulate the expression of the peptide transporters (referred to as TAP-1 and TAP-2) and major histocompatibility complex (MHC) class I in Burkitt's lymphoma (BL) cell lines. This up-regulation is often accompanied by a restoration of antigen-presenting function as measured by the ability of these cells to present endogenously expressed viral antigen to cytotoxic T lymphocytes. Here we show that the expression of LMP1 resulted in up-regulation and nuclear translocation of RelB that were coincident with increased expression of MHC class I in BL cells. Deletion of the C-terminal activator regions (CTARs) of LMP1 significantly impaired the abilities of LMP1 to translocate RelB into the nucleus and to up-regulate the expression of antigen-processing genes. Further analysis with single-point mutations within the CTARs confirmed that the residues critical for NF-kappa B activation directly contribute to antigen-processing function regulation in BL cells. This LMP1-mediated effect was blocked following expression of either dominant negative I kappa B alpha S32/36A, an NF-kappa B inhibitor, or antisense RelB. These observations indicate that upregulation of antigen-presenting function in B cells mediated by LMP1 is signaled through the NF-kappa B subunit RelB. The data provide a mechanism by which LMP1 modulates immunogenicity of Epstein-Barr virus-infected normal and malignant cells.

FIG. 1.

Schematic representation of the LMP1 constructs used in this study. Expression constructs encoding either full-length LMP1 or a series of truncated forms of LMP1 were used (A to D and H). The deletion mutant forms were designed to express a LMP1 protein in which CTAR1 and/or CTAR2 were deleted. In addition, a panel of single-point mutant forms of the LMP1-encoding gene were also used (E to G). The point mutations within the CTARs of LMP1 (PQT>AAA, Y384G, and PQT>AAA/Y384G) block TRAFF/TRADD binding and NF-κB activation. Further details of these constructs can be found in references , , and .

FIG. 2.

Regulation of RelB expression by LMP1 and its effect on antigen-presenting function. (A to D) immunohistochemical analysis of RelB expression in LMP1-negative (BJAB.gpt2) and LMP1-positive (BJAB.MTM6) cell lines. Cells (10⁵) were cytospun, fixed in paraformaldehyde, and stained with anti-RelB, followed by swine anti-rabbit Ig, and revealed with diaminobenzidine. These cells were counterstained with hematoxylin. (E) Surface HLA A1 and Bw6 expression in BLAB.gpt2 and BJAB.MTLM6 cells. BLAB.gpt2 and BJAB.MTLM6 cells were incubated with HLA allele-specific antibody, followed by fluorescein isothiocyanate-labeled anti-mouse Ig. Mean fluorescence intensity was determined by FACScalibur. (F) Endogenous processing of HLA B35-restricted EBV CTL epitopes by BJAB.gpt2 and BJAB.MTLM6 cells. BJAB.gpt2 and BJAB.MTLM6 cells were infected with either a control vaccinia virus (Vacc.TK) or a recombinant vaccinia virus encoding EBNA3. Following overnight infection, these cells were exposed to EBNA3-specific CTL clone NB26. Percent specific lysis from one of three different experiments is shown.

FIG. 3.

Role of CTARs in regulating LMP1-mediated up-regulation of surface HLA class I expression on BL cells. BJAB (A and B) and BL30 (C and D) cells were transfected with either full-length LMP1 or deletion mutant forms (del184-386, del184-351, and del231-386). At 48 h after transfection, these cells were analyzed for surface HLA expression (HLA A1, A2, or Bw6) using allele-specific antibodies as described in Materials and Methods. To precisely map the motif within the CTAR, BL30 cells were transfected with single-point mutant forms (E and F) and surface HLA expression was analyzed as described above. Data are presented as the mean ± the standard error of three different experiments.

FIG. 4.

Effects of CTARs on regulation of TAP-1 (A) and TAP-2 (B) expression in BL cells. BL30 cells were transfected with either full-length LMP1 or deletion mutant forms (del184-386, del184-351, and del231-386). At 48 h after transfection, these cells were assessed for TAP-1 and TAP-2 expression by RT-PCR using sequence-specific primers. A detailed description of the RT-PCR method used is given in Materials and Methods. Data are presented as the mean ± the standard error of five different experiments.

FIG. 5.

Immunohistochemical analysis of RelB expression in BJAB cells following transfection of either a control vector (A), full-length LMP1-B95.8 (B), del184-386 (C), or PQT>AAA/Y384G (D). Cells (10⁵) were cytospun, fixed in paraformaldehyde, and stained with anti-RelB, followed by swine anti-rabbit Ig (DAKO), and revealed with diaminobenzidine. No counterstain was used in this analysis.

FIG. 6.

Endogenous processing function in BL cells transfected with LMP1 expression vectors is dependent on the presence of the CTARs. BJAB (A) and BL30 (B) cells were transfected with the pSG5, LMP1-B95.8, del231-386, Y384G, or PQT>AAA/Y384G expression vector. At 24 h after transfection, these cells were infected with a recombinant vaccinia virus expression vector encoding EBNA3 for 14 to 16 h. After infection, these cells were used as targets in a CTL assay. For BL30 cells, an HLA B8-restricted, EBNA3-specific LC13 CTL clone was used as an effector, while for BJAB cells, HLA B35-restricted, EBNA3-specific CTL clones NB26 and NB37 were used as effector cells. Results are expressed as percent specific lysis. E:T ratio, effector-to-target cell ratio.

FIG. 7.

Effects of antisense RelB and IκBα S32/36A on the LMP1-mediated modulation of surface HLA class I expression and antigen-presenting cell function. BJAB cells were cotransfected with full-length LMP1 and antisense RelB or IκBα S32/36A, and 24 h after transfection, these cells were analyzed for surface HLA class I expression (A) and antigen-presenting cell function (B). BJAB cells cotransfected with an expression vector encoding full-length LMP1 and a control vector were used as positive controls.