Epstein-Barr virus BamHI W repeat number limits EBNA2/EBNA-LP co-expression in newly-infected B-cells and the efficiency of B-cell transformation: a rationale for the multiple W repeats in wild-type virus strains.

The genome of Epstein-Barr virus (EBV), a gamma-herpesvirus with potent B cell growth-transforming ability, contains multiple copies of a 3kb BamHIW (W) repeat sequence; each repeat carries (i) a promoter (Wp) that initiates transformation by driving EBNA-LP and EBNA2 expression, and (ii) the W1W2 exons encoding the functionally active repeat domain of EBNA-LP. A virus' W repeat number therefore influences two potential determinants of its transforming ability: the number of available Wp copies and the maximum size of the encoded EBNA-LP. Here, using recombinant EBVs, we show that optimal B cell-transformation requires a minimum of 5 W repeats; transforming ability falls progressively with viruses carrying 4, 3 and 2W repeats, as do the levels of Wp-initiated transcripts expressed early post-infection, while 1W and 0W viruses are completely non-transforming. We therefore suggest that genetic analyses of EBV transforming function should ensure that wild type and mutant strains have equal numbers (ideally at least 5) of W copies, if the analysis is not to be compromised. Attempts to enhance the transforming function of low W copy viruses, via helper EBV strains or by gene repair, suggested that the critical defect is not EBNA-LP size but the failure to achieve strong enough co-expression of EBNA-LP and EBNA2 early post-infection. We further show in ex vivo assays that EBV strains in the blood of infected individuals typically have a mean of 5-8 W copies, consistent with the view that evolution has selected for viruses with optimal transforming function.