ORIP IS ESSENTIAL FOR EBNA GENE PROMOTER ACTIVITY IN EPSTEIN-BARR VIRUS-IMMORTALIZED LYMPHOBLASTOID CELL-LINES

During Epstein-Barr virus latent infection of B lymphocytes in vitro, six viral nuclear antigens (EBNAs) are expressed from one of two promo ters, Cp or Wp, whose activities are mutually exclusive, Upon infectio n, Wp is initially active, followed by a switch to Cp for the duration of latency, In this study, the region upstream of Cp was analyzed for the presence of cis elements involved in regulating the activities of the EBNA gene promoters in established in vitro immortalized lymphobl astoid cell lines (LCLs). It was determined that oriP, the origin for episomal maintenance during latency, is essential for efficient transc ription initiation from either Cp or Wp in LCLs, as well as in some Bu rkitt's lymphoma cell lines, Deletion of the EBNA2-dependent enhancer located upstream of Cp resulted in a ca, two- to fivefold reduction in Cp activity in the LCLs assayed, More extensive deletion of sequences upstream of Cp, including the EBNA2-dependent enhancer, resulted in n early complete loss of Cp activity, This loss of activity was shown to correlate with deletion of two CCAAT boxes, a proximal CCAAT box loca ted at bp -61 to -65 and a distal CCAAT box located at bp -253 to -257 , upstream of Cp. Site-directed mutagenesis of these cis elements demo nstrated that Cp activity is highly dependent on the presence of a pro perly positioned CCAAT box, with the dependence on the distal CCAAT bo x apparent only when the proximal CCAAT box was deleted or mutated, De letion of the glucocorticoid response elements located at ca, bp -850 upstream of Cp did not result in a significant loss in activity, In ge neral, deletions which diminished Cp activity resulted in induction of Wp activity, consistent with suppression of Wp activity by transcript ional interference from Cp, The identification of oriP and the EBNA2-d ependent enhancer as the major positive cis elements involved in regul ating Cp activity in LCL suggests that EBNA gene transcription is larg ely autoregulated by EBNA 1 and EBNA 2.