Determining the role of the Epstein-Barr virus Cp EBNA2-dependent enhancerduring the establishment of latency by using mutant and wild-type viruses recovered from cottontop marmoset lymphoblastoid cell lines

Epstein-Barr virus (EBV) nuclear antigen (EBNA) 2 (EBNA2) is involved in up regulating the expression of both EBNAs and latency-associated membrane pro teins. Transcription of the six EBNA genes, which are expressed in EBV-immo rtalized primary B cells, arises from one of two promoters, Cp and Wp, loca ted near the left end of the viral genome. Wp is exclusively used to drive EBNA gene transcription during the initial stages of infection in primary B cells; induction of transcription from Cp follows. We previously have mapp ed an EBNA2-dependent enhancer upstream of Cp (M. Woisetschlaeger et at., P roc. Natl. Acad. Sci. USA 88:3942-3946, 1991) and, more recently, have demo nstrated that deletion of this enhancer results in EBV-immortalized lymphob lastoid cell lines (LCLs) that are heavily biased toward the use of Wp to d rive transcription of the EBNA genes (L. Yoo et al., J. Virol. 71:9134-9142 , 1997). To assess the immortalizing capacity of this mutant EBV and to mon itor the early events after infection of primary B cells, B cells isolated from cottontop marmosets were used to generate LCLs immortalized with the C p EBNA2 enhancer deletion mutant virus. As previously reported, all EBV-inf ected marmoset LCLs examined could be triggered to produce significant leve ls of virus. Infection of human B cells with wild-type or Cp EBNA2 enhancer mutant viruses recovered from marmoset B-cell lines demonstrated that (i) the Cp EBNA2 enhancer mutant virus immortalizes primary human B cells nearl y as efficiently as wild-type virus and (ii) the Cp EBNA2-dependent enhance r plays an important role in the induction of Cp activity during the early stages of infection. The latter is consistent with the phenotype of LCLs im mortalized with the Cp EBNA2 enhancer mutant EBV. Finally, using an establi shed LCL in which EBNA2 function is regulated by beta -estradiol, we showed that the loss of EBNA2 function results in an similar to4-fold decrease in the steady-state levels of Cp-initiated transcripts and a concomitant incr ease in the steady-state levels of Wp-initiated transcripts. Taken together , these results provide strong evidence that EBNA2 plays an important role in regulating Cp activity. These results also demonstrate that diminished i nduction of Cp activity does not appear to affect the ability of EBV to imm ortalize primary B cells in cultures. Finally, as shown here, infection of marmoset B cells with immortalization-competent mutants of EBV provides a c onvenient reservoir for the production of mutant viruses.