REGULATION OF EBNA GENE-TRANSCRIPTION IN LYMPHOBLASTOID CELL-LINES - CHARACTERIZATION OF SEQUENCES DOWNSTREAM OF BCR2 (CP)

During Epstein-Barr virus (EBV) latent infection of B lymphocytes in v itro, six EBV nuclear antigens (EBNAs) are expressed from one of two p romoters, Cp and Wp, whose activities are mutually exclusive. Upon inf ection, Wp is initially active, followed by a switch to Cp for the dur ation of latency. In this study, the impact on Cp and Wp activity of s equences downstream of the distal EBNA gene promoter, Cp, was assessed in two lymphoblastoid cell lines. Cp activity was detected in constru cts extending from just upstream of oriP to the first W1 exon. In cont rast, Wp activity required the presence of the next downstream exon, W 2. Viral sequences from -2199 to +2680 bp, relative to the Cp transcri ption start site, were dispensable for Wp activity. Sequences from +15 5 to +2680 bp, relative to the Cp transcription start site, were dispe nsable for Cp activity. Deletion of a 200-bp region from +2680 to +288 0 bp downstream of Cp decreased both Cp and Wp activity two-to fivefol d. Wp activity was also significantly diminished by deletion of the se quences from +2880 to +3000 bp downstream of the Cp transcription init iation site, which encompassed the Wp CCATT box. Based on deletion ana lyses of 10.3 kb of viral genomic sequence extending from just upstrea m of oriP to the first Wp, the only deletions which significantly upre gulated Wp activity were those which abrogated Cp activity. However, r eporter constructs in which the orientation of Cp was reversed display ed Wp activity comparable to that of reporter constructs in which Cp w as deleted, even though the steady-state level of Cp-initiated transcr ipts from the inverted promoter was indistinguishable from that observ ed with Cp in normal orientation. This is the first direct evidence to support transcriptional interference as the mechanism for the mutuall y exclusive behavior of Cp and Wp.