Continuous proliferation of EBV-transformed cells requires LMP2A. (A) The absolute numbers of B cells infected with 2089 WT EBV or 2190 EBV with its LMP2A gene flanked by 2 loxP sites were determined by FACS analysis as in Figure 2F. The cells were transduced with the E coli–derived Cre protein HTNC²³  for 3 hours, and the surviving cells were counted by FACS and set to 100% on day 1. 2089 WT EBV–infected LCLs recovered from this manipulation and started to proliferate, but LCLs infected with LMP2A⁺ 2190 EBV did not recover within the observation period. The asterisk indicates the time point when cells were removed for quantitative real-time PCR analysis of the LMP2A gene, which revealed that more than 90% of the 2190 EBV–infected cells had lost the LMP2A allele 4 days after transduction (data not shown). (B) Reverse transcription (RT)-PCR analysis of BZLF1 transcripts in HTNC-transduced 2190-infected LCLs 2 and 4 days after Cre transduction. The positions of the primer oligonucleotides P7 and P8 are schematically shown together with the predicted size of the PCR products indicative of mRNA-specific cDNA molecules of the actively transcribed BZLF1 gene and (contaminating) viral DNA. BZLF1 transcription levels did not increase but showed a slight reduction upon deletion of LMP2A, indicating that its loss did not trigger the onset of the lytic phase of EBV's life cycle.