Analysis of BHRF1 expression and CD8+ T cell recognition following EBV infection of B cells in vitro.

<p>(A) Analysis of gene expression using a 48:48 Dynamic Array QPCR assay to measure BHRF (Y2-HF, latent) and EBNA2 transcripts at time points 0.5 to 14 days post-infection with EBV (wt2089). Results are expressed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005549#ppat.1005549.g005" target="_blank">Fig 5A</a>. (B) Immunoblotting to detect expression of BHRF1 at time points between 0.5 and 7 days post EBV-infection; uninfected B cells (UI), established wild-type (WT) and BZLF1-KO LCLs and a BL cell line (Sal) were included as controls. (C) CD8+ T cell recognition of newly infected B cells. Primary B cells (HLA-A68, B*5501-positive) were infected with EBV (wt2089) then co-cultured with BHRF1 (ETF/A68) and EBNA2 (RPT/B*5501)-specific CD8+ T cell clones (40,000 B cells + 2500 T cells/well). Supernatant was harvested at the specified time points and the IFNγ concentration measured by ELISA; results are the mean of triplicate wells +/- SD. (D) BHRF1 expression during latency. BHRF1 (ETF)-specific CD8+ T cell clones were screened against a panel of HLA-matched (A68-positive) and mismatched LCL pairs; each LCL pair included the WT and BZLF1-KO LCL. IFNγ ELISA, results are shown for one representative clone. Results for T cell assays are representative of ≥2 independent assays using multiple effectors of each specificity.