Relative levels of EBNA1 gene transcripts from the C/W, F and Q promoters in Epstein-Barr virus-transformed lymphoid cells in latent and lytic stagesof infection

Four promoters, Cp, Wp, Fp and Qp, are known to participate in transcriptio n of the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) gene in EBV-inf ected cell lines. The promoters are used differentially during the differen t phases of infection and establishment of the stages of latency. This has raised questions about the regulation of the promoters and the molecular me chanisms underlying the switches between them. To obtain a measure of the a ctivity of the different EBNA1 transcription units in EBV-transformed cell lines of different phenotypes, RNA probes were constructed that allowed the detection and relative quantification, by RNase protection analysis, of EB NA1 transcripts initiated at Fp and Qp and, in an indirect manner, Cp/Wp, R Nase protection and PCR assays were performed with cytoplasmic RNA from B-l ymphoid cell lines in latency stages I, II-III and III and after induction of the virus lytic cycle. The experiments demonstrated that, in addition to previously identified EBNA1 transcripts, cell lines of all latency types a lso contained different mRNAs that carried sequences from the EBNA1-encodin g K exon, Induction of the virus lytic cycle resulted in low levels of an F pQ/U/K-spliced transcript. However, there was a large increase of FpQ- and FpQ/U-spliced transcripts with unknown 3' sequences. Furthermore, a new tra nscript, initiated at an unidentified site 5' of the BamHI f/K cleavage sit e and continuing through BamHI K into the EBNA1-encoding K exon without int erruption, was produced in substantial amounts in the lytic cycle.