A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection
Fig 8
Converting the -141 Zp nucleotide in the intact B95.8 genome to the Zp-V3 nucleotide increases lytic protein expression in stably infected Burkitt cells.
(A) EBV-negative Mutu B cells were infected with wildtype, Zp mutant, or revertant B95.8 (2089) viruses as indicated, and stably selected with hygromycin B for two months. Two different independently selected lines for each virus were then treated for two days with or without ionomycin (in the presence or absence of cyclosporine), and immunoblots were performed to detect EBNA1, EBNA2, LMP1, Z, BMRF1 (early lytic protein), p18 (late lytic protein), and actin. Kem III cell extract was included as a positive control for EBNA1, EBNA2, and LMP1. (B) Mutu cell lines containing Wt or Zp mutant viruses were nucleofected with control siRNA or NFATc1 siRNA. Ionomycin or DMSO control was added after 48 hours, and cells harvested 72 hours post-infection. Immunoblots were performed to detect NFATc1, R, BMRF1, Z, and tubulin (loading control). (C) Mutu cell lines containing Wt or Zp mutant viruses (or mock infected cells) were treated with or without anti-IgG for two days and immunoblots performed to detect BMRF1, Z, and actin (loading control). (D) Mutu cell lines containing Wt, Zp mutant, or revertant viruses were treated with or without TPA plus sodium butyrate (NaBut) (in the presence or absence of cyclosporine) for two days and immunoblots performed to detect Z expression and GAPDH (loading control). (E) ChIP assays were performed using Mutu cell lines containing Wt or Zp mutant viruses treated for three hours with ionomycin. Formaldehyde-fixed cell extracts were immunoprecipitated with control anti-IgG or NFATc1 antibody. qPCR using primers for the EBV Z promoter was performed; results shown are expressed as the amount of Zp complexed to NFATc1 ab relative to the control IgG ab. Data represent three independent experiments.