EBV-immortalized isogenic human B-cell clones exhibit differences in DNA-protein complex formation on the BZLF1 and BRLF1 promoter regions among latent, lytic and TPA-activated cell lines

Abstract

The reactivation of Epstein-Barr virus from latency requires the transcriptional induction of the viral encoded lytic cycle initiator gene, BZLF1, and a concomitant switch from OriP to OriLyt directed viral DNA replication. To investigate the role of host cell factors in these events, a series of EBV-immortalized clonal lymphoblastoid cell lines (LCL) were derived from the spontaneous outgrowth of peripheral blood lymphocytes from a single EBV-seropositive individual. We show that the state of virus activation among this family of isogenic clonal LCL differs, suggesting that each B-cell clone expresses a different complement of cellular factors that influence the state of viral activation. As a first step in the identification of factors involved in EBV reactivation, nuclear extracts were prepared from tightly latent, spontaneously replicating and latent LCL treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and sodium butyrate. The extracts were used in gel mobility shift analyses to compare DNA-protein complex formation among a series of target DNA sequences, including OriLyt and promoter sequences from BZLF1 and BRLF1. The DNA-protein complex patterns were reproducible and indistinguishable among extracts obtained from the latent LCL, but differed from those observed in extracts obtained from the spontaneously replicating LCL, particularly in regard to the binding of a CREB protein to the BZLF1 promoter. Moreover, extracts prepared from LCL treated with TPA to induce virus reactivation resulted in the formation of complexes that differed from those prepared from the spontaneously replicating LCL. Taken together, these data suggest that B-cell factors govern the state of viral activation and that EBV may be reactivated by more than one pathway.