Generation of EBV-positive 293 cell lines

293/EBV ΔBVLF1 and 293/EBV ΔBcRF1 were generated using BM2710 Escherichia coli carrying the invasin gene from Yersinia pseudotuberculosis and the hly gene encoding listeriolysin O from Listeria monocytogenes, which allow gene transfer of intact BACmids to some mammalian cells in vitro. BACmids EBV ΔBVLF1 (MI-383) and EBV ΔBcRF1 (MI-27) have been previously described as a part of a comprehensive EBV mutant library (26 (link)). BM2710 E. coli stably transformed with EBV ΔBVLF1 or EBV ΔBcRF1 was obtained from Eric Johannsen (University of Wisconsin—Madison, USA). These EBV-positive BM2710 E. coli cells were grown overnight at 32°C in brain heart infusion media (37 g/L [wt/vol]) supplemented with 0.5 mM 2,6-diaminopimelic acid (DAP) (Alfa Aesar) and 25 μg/μL spectinomycin (Alfa Aesar) and selected with 50 μg/mL kanamycin. One milliliter of BM2710 E. coli overnight culture was added to 293 cells at 80 to 90% confluence in a 60-mm cell culture plate, in 1× DMEM supplemented with 0.5 mM DAP and 25 μg/μL spectinomycin, and incubated for 2 h. Following incubation, the medium was removed, the cells were washed with 1× DMEM to remove as many bacteria as possible, and fresh 1× DMEM plus 10% FBS medium was added, supplemented with 50 μg/mL gentamicin (Gibco). The following day, the medium was replaced with 1× DMEM plus 10% FBS. EBV-positive 293 cells were selected and maintained with 200 μg/mL hygromycin B.