Control of Epstein-Barr virus reactivation by activated CD40 and viral latent membrane protein 1

Abstract

In humans, Epstein-Barr virus (EBV) establishes a persistent latent infection in peripheral resting B lymphocytes. Virus reactivation is highly restricted. Whereas in healthy humans the infection usually is benign, immunocompromised patients show an increased risk for EBV-associated malignancies, accompanied by an increase in virus replication and in the number of virus-infected cells. To search for viral and host factors regulating virus reactivation, we used conditionally EBV-immortalized B cells. We found that CD40-CD40 ligand interaction and the viral mimic of activated CD40, EBV latent membrane protein 1, suppress virus reactivation. Both inhibit anti-IgM or phorbolester-induced transcription of the viral immediate early protein BZLF1, which controls entry into the viral lytic cycle. The finding that latent membrane protein 1 and CD40 contribute to the regulation of latency may have important implications for the balance between EBV and its host in normal as well as in immunocompromised individuals.

Figure 1

EBV reactivation in the presence and absence of active EBNA2. EREB2–1 cells incubated in medium containing estrogen (plus estrogen; EBNA2 is active) or deprived of estrogen for 24 h (minus estrogen; EBNA2 is inactive) were plated on 24-well plates at a density of 2.5 × 10⁵ cells/ml. Cells were stimulated by transfer into wells precoated with anti-human IgM (5 μg/well) or addition of 20 ng/ml PMA or were left untreated. Cells were harvested 24 (A) or 72 (B) h after stimulation, intracellularly stained for (A) BZLF1 or (B) VCA expression, and analyzed by FACS analysis. Data shown are means of triplicates ± SD of three independent experiments.

Figure 2

LMP1 can inhibit EBV reactivation in the absence of EBNA2. Untransfected cells, cells transfected with the vector control (pHEBo-1A), and stably transfected EREB2–5 cells constitutively expressing LMP1 (SVLMP1–13C and SVLMP1–11C) were tested for virus reactivation in the absence of estrogen. Cells were stimulated with PMA or plate-bound anti-human IgM for 72 h. Virus reactivation was examined by intracellular staining for VCA expression and FACS analysis. Data shown are means ± SD of three independent experiments.

Figure 3

CD40 ligand can inhibit EBV reactivation. (A) A total of 2.5 × 10⁵ EREB 2–1 cells deprived of estrogen were stimulated with PMA or anti-IgM in the absence or presence of increasing numbers of irradiated CD40 ligand-expressing LTK⁻ cells (50 ×, 150 ×, and 250 × 10³ cells) or LTK⁻ cells as a control. Seventy two hours after stimulation cells were stained for intracellular VCA expression and analyzed by FACS. Data shown are means of three independent experiments ± SD. Values marked by * are significantly different from those of cells incubated without LTK⁻ cells (P < 0.05, unpaired Student's t test). For cocultivation with 150 × and 250 × 10³ CD40 ligand-expressing cells and anti-IgM stimulation the SD values fall within the symbol. (B) A total of 2.5 × 10⁵ Akata cells were mixed with 2.5 × 10⁵ CD40 ligand-expressing LTK⁻ cells or LTK⁻ control cells, stimulated with plate-bound anti-IgG for 72 h and stained for VCA expression by intracellular FACS analysis. Data shown are means of three independent experiments ± SD. The data marked by * are significantly different from those of cells incubated without LTK⁻ cells (P < 0.05, unpaired Student's t test). (C) Immunoblot of early antigen (EA) expression in Mutu I and Elijah cells after anti-IgM stimulation. A total of 2.5 × 10⁵ Mutu I or Elijah cells were stimulated with plate-bound anti-IgM and cocultivated with 2.5 × 10⁵ irradiated CD40 ligand-expressing LTK⁻ cells or LTK⁻ cells as a control. Forty eight hours after stimulation cells were harvested for preparation of cell extracts and analyzed by immunoblot using a human antiserum reactive with EBV early antigens. The positions of the molecular mass standards (kDa) are indicated on the left.

Figure 4

CD40 ligand inhibits BZLF1 expression in a dose-dependent fashion. Immunoblot of BZLF1 expression in EREB2–1 cells after anti-IgM and CD40 ligand activation. A total of 2.5 × 10⁵ cells cultured in estrogen-free medium were stimulated with plate-bound anti-IgM or PMA in the absence or presence of increasing numbers of irradiated CD40 ligand-expressing LTK⁻ cells (10 ×, 50 ×, 150 ×, and 250 × 10³ cells) or LTK⁻ cells as a control. Twenty-four hours after stimulation, cells were harvested for preparation of cell extracts. To adjust for comparable protein concentrations of cell extracts, LTK⁻ cells were added to a final concentration of 2.5 × 10⁵ LTK⁻ cells directly before preparation of the extracts.

Figure 5

LMP1 and activated CD40 inhibit BZLF1 transcription. PHEBo-1A (stably pHEBo-transfected), SVLMP1–11C (stably SVLMP1-transfected), and untransfected EREB2–1 cells were incubated without estrogen for 24 h and then stimulated with PMA. PHEBo-1A and SVLMP1–11C cells were stimulated for 0, 4, and 16 h and EREB2–1 cells for 16 h in the absence or presence of irradiated CD40 ligand-expressing LTK⁻ cells or LTK⁻ cells as controls. BZLF1 expression was monitored by Northern blot analysis using total cellular RNA and a labeled 0.9-kb BZLF1 probe. The positions of the 1.0-kb BZLF1 and the 2.8-kb BRLF1/BZLF1 transcripts are indicated by arrows. The 28S and 18S bands of the ethidium bromide-stained RNA gels served as loading controls.