MicroRNAs have been implicated in the modulation of gene expression programs important for normal and cancer cell development. miR-155 is known to play a role in B-cell development and is upregulated in various B-cell lymphomas, including several that are latently infected with Epstein-Barr virus (EBV). We show here that EBV infection of primary human B lymphocytes leads to the sustained elevation of miR-155 and its precursor RNA, BIC. The EBV-encoded latency membrane protein 1 (LMP1) can partially reconstitute BIC activation in B lymphocytes but not in epithelial cell cultures. LMP1 is a potent activator of NF-κB signaling pathways and is essential for EBV immortalization of B lymphocytes. An inhibitor to miR-155 further stimulated NF-κB responsive gene transcription, and IKKε was identified as a potential target of miR-155 translational repression. Remarkably, miR-155 inhibitor reduced EBNA1 mRNA and the EBV copy number in latently infected cells. This suggests that miR-155 contributes to EBV immortalization by modulation of NF-κB signaling and the suppression of host innate immunity to latent viral infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a cluster of 12 microRNAs (miRNAs) that are processed from a transcript that is embedded within the major latency control region. We have generated a deletion mutation that eliminates 10 of the 12 viral miRNAs from the KSHV bacmid by using recombineering methods. The KSHV miRNA deletion mutant (BAC36 ΔmiR) behaved similarly to wild-type (wt) BAC36 in viral production, latency gene transcription, and viral DNA copy number in 293 and dermal microvascular endothelial cells (DMVECs). However, BAC36 ΔmiR consistently expressed elevated levels of viral lytic genes, including the immediate-early transcriptional activator Rta (ORF50). At least one KSHV microRNA (miRK12-5) was capable of suppressing ORF50 mRNA, but poor seed sequence alignments suggest that these targets may be indirect. Comparison of epigenetic marks in ΔmiR KSHV genomes revealed decreases in histone H3 K9 methylation, increases in histone H3 acetylation, and a striking loss of DNA methylation throughout the viral and cellular genome. One viral miRNA, K12-4-5p, was found to have a sequence targeting retinoblastoma (Rb)-like protein 2 (Rbl2), which is a known repressor of DNA methyl transferase 3a and 3b mRNA transcription. We show that ectopic expression of miR-K12-4-5p reduces Rbl2 protein expression and increases DNMT1, -3a, and -3b mRNA levels relative to the levels for control cells. We conclude that KSHV miRNA targets multiple pathways to maintain the latent state of the KSHV genome, including repression of the viral immediate-early protein Rta and a cellular factor, Rbl2, that regulates global epigenetic reprogramming.