The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis

- The EBV miR-BHRF1 microRNAs do not up upregulate B cell growth in trans.
- EBNA-LP expression is downregulated by pri-miR-BHRF1-2 and 1-3 acting in cis.
- Loss of miR-BHRF1-2 and 1-3 causes EBNA-LP overexpression and inhibits B cell growth.
- Novel alternative splicing of EBV Cp/Wp transcripts was identified.

The Epstein-Barr virus (EBV) miR-BHRF1 microRNA (miRNA) cluster has been shown to facilitate B-cell transformation and promote the rapid growth of the resultant lymphoblastoid cell lines (LCLs). However, we find that expression of physiological levels of the miR-BHRF1 miRNAs in LCLs transformed with a miR-BHRF1 null mutant (∆123) fails to increase their growth rate. We demonstrate that the pri-miR-BHRF1-2 and 1-3 stem-loops are present in the 3'UTR of transcripts encoding EBNA-LP and that excision of pre-miR-BHRF1-2 and 1-3 by Drosha destabilizes these mRNAs and reduces expression of the encoded protein. Therefore, mutational inactivation of pri-miR-BHRF1-2 and 1-3 in the ∆123 mutant upregulates the expression of not only EBNA-LP but also EBNA-LP-regulated mRNAs and proteins, including LMP1. We hypothesize that this overexpression causes the reduced transformation capacity of the ∆123 EBV mutant. Thus, in addition to regulating cellular mRNAs in trans, miR-BHRF1-2 and 1-3 also regulate EBNA-LP mRNA expression in cis.