Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation

MicroRNAs (miRNAs) are post-transcriptional regulatory RNAs that can modulate cell signaling and play key roles in cell state transitions. Epstein-Barr virus (EBV) expresses >40 viral miRNAs that manipulate both viral and cellular gene expression patterns and contribute to reprogramming of the host environment during infection. Here, we identified a subset of EBV miRNAs that desensitize cells to B cell receptor (BCR) stimuli, and attenuate the downstream activation of NF-kappaB or AP1-dependent transcription. Bioinformatics and pathway analysis of Ago PAR-CLIP datasets identified multiple EBV miRNA targets related to BCR signal transduction, including GRB2, SOS1, MALT1, RAC1, and INPP5D, which we validated in reporter assays. BCR signaling is critical for B cell activation, proliferation, and differentiation, and for EBV, is linked to reactivation. In functional assays, we demonstrate that EBV miR-BHRF1-2-5p contributes to the growth of latently infected B cells through GRB2 regulation. We further determined that activities of EBV miR-BHRF1-2-5p, EBV miR-BART2-5p, and a cellular miRNA, miR-17-5p, directly regulate virus reactivation triggered by BCR engagement. Our findings provide mechanistic insight into some of the key miRNA interactions impacting the proliferation of latently infected B cells and importantly, governing the latent to lytic switch. Author summary Understanding the molecular mechanisms regulating EBV latency and entry into the lytic replication cycle is important in developing therapies for viral disease. We demonstrate here that a subset of EBV miRNAs target host factors within the BCR signaling pathway and consequently, negatively regulate cellular responses to BCR cross-linking. Disrupting activity of individual EBV miRNAs, specifically miR-BHRF-1-2-5p and miR-BART2-5p, enhanced lytic gene expression following BCR engagement, suggesting a key role for these miRNAs in attenuating viral reactivation. Our experiments establish a link between EBV miRNAs and signaling through the BCR, providing new insight into miRNA-mediated aspects of latency and reactivation.