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Epstein-Barr virus LMP2A suppresses MHC class II expression by regulating the B-cell transcription factors E47 and PU.1

Jiun-Han Lin, Ju-Yin Lin, Ya-Ching Chou, Mei-Ru Chen, Te-Huei Yeh, Chung-Wu Lin, Sue-Jane Lin and Ching-Hwa Tsai

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Article Figures & Data

Figures

  • Figure 1

    Downregulation of the expression of MHC class II and CD74 in LCLs. CD19-positive B cells were seeded in a 12-well plate at a density of 1 × 106 cells per well and infected with EBV. RNA and proteins were harvested at the time points indicated. (A) The expression of HLA-DRA, HLA-DRB1, and CD74 transcripts was measured by RT-Q-PCR. The relative fold of the transcripts was normalized to uninfected B cells with the corresponding β-actin mRNA. This is a representative result from 6 independent experiments from anonymous donors. (B) Protein expression of MHC class II, CD74, EBNA1, LMP2A, and β-actin was detected by western blotting. Detection of β-actin served as an internal control. (C) BJAB cells were transfected with EBNA1 or BARF0 expression plasmids or infected with EBER1, EBER2, LMP1, LMP2A, or Zta lentiviruses. Expression of MHC class II, CD74, EBNA1, EBER1, EBER2, BARF0, LMP1, LMP2A, and Zta transcripts in the transfectants and lentiviruses-infected cells were analyzed by RT-PCR. β-actin was detected as an internal control. (D) Akata and BJAB cells were infected with LMP2A-expressing lentiviruses. Cell lysates were harvested and the expression of MHC class II, CD74, and LMP2A was detected by western blotting. β-actin was detected as an internal control. (E) BJAB cells were infected with various doses of LMP2A-expressing lentiviruses. At day 5 postinfection, cell lysates were harvested for the detection of MHC class II, CD74, and LMP2A by western blot analysis. β-actin was detected as an internal control. The experiment was performed 3 times, and 1 representative is shown.

  • Figure 2

    LMP2A downregulates the expression of CIITA, E47, and PU.1. (A) Akata and BJAB cells were infected with LMP2A-expressing lentiviruses. Expression of CIITA and LMP2A was detected by western blotting. (B) The expression of the CIITA-PI, CIITA-PIII, CIITA-PIV, and LMP2A transcripts was measured by RT-PCR analysis. Raji cells were used as a positive control for the CIITA promoters. (C) Expression of E47, PU.1, IRF4, IRF8, and LMP2A was measured by RT-PCR analysis. (D) Expression of E47, PU.1, and LMP2A was detected by western blotting. (E) Akata and BJAB cells were infected with Zta-expressing lentiviruses. Expression of E47, PU.1, and Zta was detected by western blotting. β-actin was detected as an internal control. Each experiment was performed 3 times, and 1 representative is shown.

  • Figure 3

    LMP2A downregulates the expression of E47 and PU.1 through the PI3K/Akt pathway. (A) BJAB cells were infected with LMP2A-, LMP2A-Δ74-85–, or LMP2A-Y112F–expressing lentiviruses. Expression of LMP2A, E47, PU.1, CD74, and MHC class II was analyzed by western blotting. β-actin was detected as an internal control. (B) BJAB cells were transfected with wild-type LMP2A or LMP2A with mutated PY motif expression plasmids, and the transfectants were subjected to western analysis of LMP2A, E47, and PU.1. β-actin was detected as an internal control. (C) LCLs were cultured in the presence of 25 μM Piceatannol or 10 μM PP2 at the indicated time. Expression of phospho-Akt (pAkt), total Akt, E47, and PU.1 was analyzed by western blotting. β-actin was detected as an internal control. (B-C) Experiments were performed twice, and 1 representative is shown. (D-E) BJAB cells were infected with LMP2A-expressing lentiviruses or vector control. After 5 days, the cells were cultured in the presence of 20 μM LY294002 (D) or 20 μM U0126 (E) paired to dimethylsulfoxide (DMSO) control for 48 hours. Cell lysates were subjected to western analysis of E47, PU.1 CD74, MHC class II, and LMP2A. β-actin was detected as an internal control. (D) Special detection of pAkt and total Akt and (E) detection of phosphoERK1/2 (pERK1/2) and total ERK. (F) LCLs were infected with sh-Luc or sh-PIK3CA expressing lentivirus and the cell lysates were subjected to western analysis of E47, PU.1, CD74, and MHC class II. β-actin was detected as an internal control. (G) BJAB cells were infected with LMP2A, PU.1, or forced-dimered-E47 (FD-E47) lentiviruses and transfectants were subjected to western analysis of FD-E47, endogenous E47 (endo-E47), PU.1, CD74, MHC class II, and LMP2A. β-actin was detected as an internal control. Each experiment was performed 3 times, and 1 representative is shown.

  • Figure 4

    LMP2A inhibited the E47 and PU.1 promoter activity through the ITAM motif. (A) Akata and BJAB cells were infected with pSIN, LMP2A, LMP2A-Y112F, and LMP2A-Δ74-85 expressing lentiviruses. After 3 days, the cells were infected with pCDH-GL3–, E47-, and PU.1 reporter–expressing lentiviruses. Luciferase activities were normalized with the GFP intensities of each transfectant. The activated fold was calculated by normalizing luciferase activities for the transfectant vs that for the pSIN with pCDH-GL3 vector control. (B) BJAB cells were infected with pSIN- and LMP2A-expressing lentiviruses. After 3 days, the cells were infected with pCDH-GL3–, E47-, and PU.1-expressing lentiviruses and incubated with dimethylsulfoxide (DMSO) or 20 μM LY294002 for another 48 hours. Luciferase activities from each transfectant were normalized with the GFP intensities. The activated fold for each reporter was calculated by normalizing luciferase activities for the transfectant vs that for the pSIN with pCDH-GL3 vector control. These data are a composite of 3 independent experiments (mean ± standard deviation).

  • Figure 5

    LMP2A inhibited the E47 and PU.1 promoter activity. (A) Schematic map of the promoter region of PU.1 (−470 to +57). Deletion constructs derived from the PU.1 construct (−470 to +57) were subcloned into the pCDH-GL3 luciferase reporter vector. BJAB cells were infected with pSIN- and LMP2A-expressing lentiviruses. After 3 days, the cells were infected with the reporter lentiviruses indicated. After 4 days, luciferase activities from each transfectant were normalized with the GFP intensities. (B) BJAB cells were transduced with pLKO or PU.1 lentiviruses. After 5 days, infected BJAB cells were transduced with PU.1- (−470 to +57), pSIN-, or LMP2A-expressing lentiviruses and then the luciferase activities were measured and normalized to GFP activity at day 5 postinfection. (C) BJAB cells were transduced with shLuc or shPAX5 lentiviruses. After 5 days, infected BJAB cells were further transduced with PU.1- (−470 to +57), pSIN-, or LMP2A-expressing lentiviruses. At day 5 postinfection, the luciferase activities were measured and normalized to GFP activity. (D) Schematic map of the promoter region of E47 (−1000 to +29). Deletion constructs derived from the E47 construct (−1000 to +29) were subcloned into the pCDH-GL3 luciferase reporter vector. The 293T cells were transfected with pSG5- and LMP2A-expressing plasmids. After 3 days, the luciferase activities were measured and normalized to GFP activity. (E) The 293T cells were transfected with pCDH-GL3-E47– (−1000 to +29), pSG5-, or LMP2A-expressing plasmids and treated with 500 nM Mithramycin. At day 3 postinfection, the luciferase activities were measured and normalized to GFP activity. (F) The 293T cells were infected with shLuc or shSP1 lentiviruses. After 5 days, infected 293T cells were transfected with pCDH-GL3-E47– (−1000/+29), pSG5-, or LMP2A-expressing plasmids. At day 3 postinfection, the luciferase activities were measured and normalized to GFP activity. Each experiment was performed 3 times, and 1 representative is shown.

  • Figure 6

    LMP2A is the key factor of EBV inhibiting the expression of E47 and PU.1 and their downstream genes. (A) Expression of EBNA1, LMP1, LMP2A, E47, PU.1, and β-actin in various pairs of uninfected primary B cells and EBV-immortalized LCLs was detected by western blotting. β-actin served as an internal control. (B-H) The LCL lines B36, B37, B44, and B47 were infected with shLuc or shLMP2A lentiviruses. After 5 days, the RNA and protein were harvested. Expression levels of the LMP2A (B), E47 (C), PU.1 (D), CIITA (E), HLA-DRA (F), HLA-DRB (G), and CD74 (H) transcripts were measured by RT-Q-PCR. (I) LCL B47 cells were infected with PU.1 or FD-E47 expression lentiviruses. Expression of E47, PU.1, CD74, MHC class II, and LMP2A was analyzed by western blotting. β-actin was detected as an internal control. (Paired t test, *P < .05; **P < .01). Each experiment was performed 3 times, and 1 representative is shown.

  • Figure 7

    HLA-DR is not expressed in PTLD biopsies compared with tonsil biopsies. PTLD, tonsils, and HD sections were used for IHC assays and the nuclei counterstained with hematoxylin. (A) Positive signals of HLA-DR were observed as a brown color in tonsil biopsies but not in PTLD biopsies. (B) Positive signals of HLA-DR were observed as a brown color in EBER− biopsy (case 11) but not in EBER+ biopsy (case 1). The nuclei are stained blue. Original magnification, ×200. Scale bar indicates 50 μm.