Latent Membrane Protein 2B Regulates Susceptibility to Induction of Lytic Epstein-Barr Virus Infection

TLR9 triggering in Burkitt's lymphoma cell lines suppresses the EBV BZLF1 transcription via histone modification

“…Calcium-flux measurements and flow cytometry Cells (1 Â 10 7 /ml À1 ) were treated with CpG2006, followed by Fluo-3 and Fura-Red Dyes final concentrations of 6 and 15 mM, respectively, Invitrogen, Basel, Switzerland) and antiIgG before measurement with a FC-500 (Beckmann-Coulter, Fullerton, CA, USA) for 5 min as a ratio of Fluo-3/Fura Red as described (Novak and Rabinovitch, 1994;Rechsteiner et al, 2008).…”

“…Cell culture and induction of lytic EBV Akata and Mutu I BL cells were cultured as described (Rechsteiner et al, 2008). Induction of lytic EBV was accomplished with 100 mg/ ml anti-IgG (Dako, DakoCytomation, Zug, Switzerland), 10 mg/ ml anti-IgM (Dako) or 5 ng/ml human TGFb (R&D Systems, Wiesbaden-Nordenstadt, Germany).…”

“…Digestion with SmaI and NotI resulted in the isolation of the FLAG-MyD88lpr, which was cloned into the polylinker site of the pEneo vectors (Rechsteiner et al, 2007) through PmeI and NotI. Electroporation and selection were performed as described (Rechsteiner et al, 2008).…”

“…RNA preparation, reverse transcription and qPCR (TaqMan) RNA isolation, DNAse treatment, reverse transcription and qPCR (TaqMan) was performed as described before (Ladell et al, 2007;Rechsteiner et al, 2008). BZLF1, BRLF1, BXLF1, BXLF2, BCRF1, MyD88wt, MyD88mutant or hIL10 mRNA expression was normalized to the housekeeping genes mRNA of HMBS, GAPDH or 18sRNA, resulting in D cycle threshold (DC T ) values.…”

“…Therefore, CpG ODN treatment could possibly affect levels of Lyn (Sanjuan et al, 2006), leading to an inhibition of the Ca 2 þ flux, and thus reduce BZLF1 mRNA expression. However, when we triggered TLR9 before BCR crosslinking, the signal for Ca 2 þ release, measured by flow cytometry (Novak and Rabinovitch, 1994;Rechsteiner et al, 2008), was as strong as after BCR crosslinking alone (Figure 2a). Thus, TLR9 triggering does not interfere with BCR crosslinking itself or with initial steps of BCR signaling leading to Lyn activation and Ca 2 þ release.…”