A simple reverse transcriptase PCR assay to distinguish EBNA1 gene transcripts associated with type I and II latency...

In Epstein-Barr virus (EBV)-associated tumors that arise in immunocompetent individuals, the pattern of viral gene expression is very restricted compared with that of latently infected B cells in tissue culture. A hallmark of viral gene expression in these tumors is the exclusive expression of only one EBV-encoded nuclear antigen, EBNA1, which is driven from a promoter (Qp) that lies near the junction of the viral BamHI F and Q fragments. During induction of the lytic cycle, a viral promoter, Fp, which lies ca. 200 bp upstream of Qp, gives rise to transcripts which overlap with Qp-initiated EBNA1 gene transcripts. Distinguishing between latency-associated EBNA1 gene transcripts and those associated with the early phase of the viral lytic cycle is critical for correct identification of restricted viral latency. Here we describe a reverse transcriptase PCR protocol which employs a nested set of upstream primers from the BamHI Q region of the viral genome and readily distinguishes Fp-initiated transcripts from Qp-initiated transcripts. A single set of amplification conditions was used for the various PCR primer combinations, which allowed all reactions to be run simultaneously. An in vitro-generated transcript, diluted in RNA from an EBV-negative cell line, was used to demonstrate that the efficiencies of amplification with the different primer combinations were very similar. This protocol was used to demonstrate that EBNA1 gene transcription in two previously uncharacterized EBV-positive epithelial cell lines initiates from Qp. In addition, we assessed the site(s) of initiation of EBNA1 gene transcripts in cell lines exhibiting restricted viral latency. Contrary to the results of Nonkwelo et al. (J. Virol. 70:623-627, 1996), which indicated that EBNA1 gene transcription during restricted viral latency initiates at multiple sites downstream of Fp, we show here that nearly all EBNA1 transcripts start at the previously identified Qp transcription initiation site.