A Viral microRNA Cluster Regulates the Expression of PTEN, p27 and of a bcl-2 Homolog

The Epstein-Barr virus (EBV) infects and transforms B-lymphocytes with high efficiency. This process requires expression of the viral latent proteins and of the 3 miR-BHRF1 microRNAs. Here we show that B-cells infected by a virus that lacks these non-coding RNAs (Δ123) grew more slowly between day 5 and day 20, relative to wild type controls. This effect could be ascribed to a reduced S phase entry combined with a moderately increased apoptosis rate. Whilst the first phenotypic trait was consistent with an enhanced PTEN expression in B-cells infected with Δ123, the second could be explained by very low BHRF1 protein and RNA levels in the same cells. Indeed, B-cells infected either by a recombinant virus that lacks the BHRF1 protein, a viral bcl-2 homolog, or by Δ123 underwent a similar degree of apoptosis, whereas knockouts of both BHRF1 microRNAs and protein proved transformation-incompetent. We find that that the miR-BHRF1-3 seed regions, and to a lesser extent those of miR-BHRF1-2 mediate these stimulatory effects. After this critical period, B-cells infected with the Δ123 mutant recovered a normal growth rate and became more resistant to provoked apoptosis. This resulted from an enhanced BHRF1 protein expression relative to cells infected with wild type viruses and correlated with decreased p27 expression, two pro-oncogenic events. The upregulation of BHRF1 can be explained by the observation that large BHRF1 mRNAs are the source of BHRF1 protein but are destroyed following BHRF1 microRNA processing, in particular of miR-BHRF1-2. The BHRF1 microRNAs are unlikely to directly target p27 but their absence may facilitate the selection of B-cells that express low levels of this protein. Thus, the BHRF1 microRNAs allowed a time-restricted expression of the BHRF1 protein to innocuously expand the virus B-cell reservoir during the first weeks post-infection without increasing long-term immune pressure.