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During Epstein-Barr virus (EBV) latent infection of B lymphocytes in vitro, six EBV nuclear antigens (EBNAs) are expressed from one of two promoters, Cp and Wp, whose activities are mutually exclusive. Upon infection, Wp is initially active, followed by a switch to Cp for the duration of latency. In this study, the impact on Cp and Wp activity of sequences downstream of the distal EBNA gene promoter, Cp, was assessed in two lymphoblastoid cell lines. Cp activity was detected in constructs extending from just upstream of oriP to the first W1 exon. In contrast, Wp activity required the presence of the next downstream exon, W2. Viral sequences from -2199 to +2680 bp, relative to the Cp transcription start site, were dispensable for Wp activity. Sequences from +155 to +2680 bp, relative to the Cp transcription start site, were dispensable for Cp activity. Deletion of a 200-bp region from +2680 to +2880 bp downstream of Cp decreased both Cp and Wp activity two- to fivefold. Wp activity was also significantly diminished by deletion of the sequences from +2880 to +3000 bp downstream of the Cp transcription initiation site, which encompassed the Wp CCATT box. Based on deletion analyses of 10.3 kb of viral genomic sequence extending from just upstream of oriP to the first Wp, the only deletions which significantly upregulated Wp activity were those which abrogated Cp activity. However, reporter constructs in which the orientation of Cp was reversed displayed Wp activity comparable to that of reporter constructs in which Cp was deleted, even though the steady-state level of Cp-initiated transcripts from the inverted promoter was indistinguishable from that observed with Cp in normal orientation. This is the first direct evidence to support transcriptional interference as the mechanism for the mutually exclusive behavior of Cp and Wp.

Regulation of EBNA gene transcription in lymphoblastoid cell lines: characterization of sequences downstream of BCR2 (Cp)