BCR⁺ as well as BCR^- B cells infected with EBV enter the S phase of the cell cycle and proliferate in vitro. (A) An EBV genome, which encodes GFP,¹⁷  was used to infect the BCR^- and BCR⁺ cell populations (middle and bottom panels) or BCR⁺ cells were left uninfected (top row panels). GFP⁺ cells were found in BCR⁺ and BCR^- cells, indicating that EBV can infect cells in both populations. (B) To analyze the cell cycle distribution of the uninfected and infected BCR^- and BCR⁺ bulk populations, the cells were labeled with the nucleotide analog BrdU 4 days after infection for 2 hours. Dual parameter flow cytometry of the cells with an APC-conjugated α-BrdU antibody and with 7-AAD to reveal the cellular DNA content indicated that the EBV-infected B-cell pool entered S phase and proliferated irrespective of their BCR surface status (middle and bottom panels, gates R2 and R3). Cells in the uninfected population enter neither S phase (top panel, region R2) nor M phase (region R3), as indicated by the lack of BrdU incorporation and predominance of G1/G0 phase (gate R1). (C) Dual parameter flow cytometry of EBV-infected B cells gated for typical forward/sideward scatter lymphocyte morphology. (D) Triple parameter flow cytometry of GFP⁺/EBV-infected B cells (gate M1 in panel A). BrdU incorporation and 7-AAD staining showed a similar cell cycle distribution in panels B, C, and D. Since only a fraction of EBV-infected primary B cells express GFP at a detectable level, the number of GFP-positive cells in the BCR⁺ and BCR^- populations is markedly lower than in panel C. Also in contrast to panel B, most GFP⁺ cells in D are not apoptotic, as indicated by a minority of cells with sub-G1 DNA content.