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. 2011 Aug;85(15):7622–7633. doi: 10.1128/JVI.00304-11

Fig. 7.

Fig. 7.

Analysis of episome persistence at early time points indicates that the internal LANA domains exert a critical role. Control BJAB cells and BJAB cells stably expressing the different LANA constructs were transfected with p8TR and cultured as described in the legend to Fig. 6. Gardella cell analyses to detect the presence of episomal p8TR were performed at the indicated time points. Samples were resolved in 0.8% agarose gel followed by Southern blotting. DNA was detected using a 32P-labeled TR fragment probe, and the blot was exposed to film for the indicated times. In order to detect smaller amounts of DNA in control BJAB and in cells expressing LANA or LANA deletion mutants, twice as many cells were loaded per well (2 × 106 cells) in Gardella gels at 12 days under selection and onwards. Naked plasmid p8TR DNA was loaded in indicated lanes as a control. (A) Episomal p8TR DNA at 24 h posttransfection. BCBL-1 (lane 1), p8TR plasmid (lane 2), BJAB cells (lane 3), BJAB cells expressing LANA (lane 4), LANAΔ465–497 (lane 5), LANAΔ33–888 (lane 6), LANAΔ33–899 (lane 7), LANAΔ33–929 (lane 8), or LANAΔ33–949 (lane 9). (B) Episomal p8TR DNA at 72 h posttransfection. BCBL-1 (lane 1), p8TR (lane2), BJAB (lane 4), BJAB cells expressing LANA (lane 5), LANAΔ465–497 (lane 6), LANAΔ33–888 (lane 7), LANAΔ33–899 (lane 8), LANAΔ33–929 (lane 9), or LANAΔ33–949 (lane 10). (C) Episomal p8TR DNA at 4 days under G418 selection (lanes 4 through 10) and 6 days under G418 selection (lanes 11 through 17). Pellets from cells at 4 days under G418 selection were frozen until the day of the Gardella analysis at 6 days under selection. BCBL-1 (lane 1), p8TR (lane 2), lane 3 is empty, control BJAB cells (lanes 4 and 11), and BJAB cells expressing LANA (lanes 5 and 12), LANAΔ465–497 (lanes 6 and 13), LANAΔ33–888 (lanes 7 and 14), LANAΔ33–899 (lanes 8 and 15), LANAΔ33–929 (lanes 9 and 16), and LANAΔ33–949 (lanes 10 and 17). (D) Episomal p8TR DNA at 8 days under G418 selection (lanes 4 through 10) and 10 days under G418 selection (lanes 11 through 17). Pellets from cells at 8 days under G418 selection were frozen until the day of the Gardella analysis at 10 days under selection. BCBL-1 (lane 1), p8TR (lane 2), lane 3 is empty, control BJAB cells (lanes 4 and 11), and BJAB cells expressing LANA (lanes 5 and 12), LANAΔ465–497 (lanes 6 and 13), LANAΔ33–888 (lanes 7 and 14), LANAΔ33–899 (lanes 8 and 15), LANAΔ33–929 (lanes 9 and 16), and LANAΔ33–949 (lanes 10 and 17). (E) Episomal p8TR DNA at 12 days under G418 selection (lanes 4 through 10) and 14 days under G418 selection (lanes 11 through 17). Pellets from cells at 12 days under G418 selection were frozen until the day of the Gardella analysis at 14 days under selection. BCBL-1 (lane 1), lane 2 is empty, p8TR (lane 3), control BJAB cells (lanes 4 and 11), and BJAB cells expressing LANA (lanes 5 and 12), LANAΔ465–497 (lanes 6 and 13), LANAΔ33–888 (lanes 7 and 14), LANAΔ33–899 (lanes 8 and 15), LANAΔ33–929 (lanes 9 and 16), and LANAΔ33–949 (lanes 10 and 17). The panel on the right is the same blot but is after a 3-day exposure to film to better show episomal p8TR in the LANA deletion mutants. (F) Episomal p8TR DNA at 16 days under G418 selection (lanes 4 through 10) and 18 days under G418 selection (lanes 11 through 17). Pellets from cells at 16 days under G418 selection were frozen until the day of the Gardella analysis at 18 days under selection. BCBL-1 (lane 1), p8TR (lane 2), lane 3 is empty, control BJAB cells (lanes 4 and 11), and BJAB cells expressing LANA (lanes 5 and 12), LANAΔ465–497 (lanes 6 and 13), LANAΔ33–888 (lanes 7 and 14), LANAΔ33–899 (lanes 8 and 15), LANAΔ33–929 (lanes 9 and 16), and LANAΔ33–949 (lanes 10 and 17). This figure is representative of two experiments.