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

Abstract

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.

HCMV miR-US22 down-regulation of EGR-1 regulates CD34+ hematopoietic progenitor cell proliferation and viral reactivation

“…HCMV miR-UL148D was also reported to facilitate HCMV latency by inhibiting immediate early response gene 5 that promotes cell division cycle 25B protein and cyclin-dependent kinase 1-mediated suppression of IE72 [50]. EBV miRNAs were recently shown to regulate B Cell receptor signaling [51], and thus B cell activation, proliferation, and differentiation [52]. In this study, EBV miR-BHRF1-2-5p promotes latency by targeting GRB2 that is part of a signaling cascade that activates transcription factors, such as NFκB and Jun that induce genes that participate in B cell proliferation and survival.…”

B Cell Receptor-Responsive miR-141 and viral miR-BART9 promote Epstein-Barr Virus Reactivation Through FOXO3 Inhibition

“…psiCheck2 3’UTR constructs for FOXO3, RANBP9, and YY1 were provided by Dr. Jay Nelson’s laboratory at VGTI. Additional 3’UTR reporters are cloned into pLSG as previously described (31, 47). Oligonucleotide sequences used for cloning are available upon request.…”

“…For viral loads, genomic DNA was isolated using DNAzol (Thermofisher). 100 ng of DNA were analyzed using primers to the LMP1 region and normalized to GAPDH levels as previously described (31). All PCR reactions were performed in technical replicates (duplicates or triplicates).…”

“…However, viral miRNA expression is rapidly induced upon entry into the lytic cycle (27, 28), indicating a possible role in EBV reactivation. Recently, we identified several EBV miRNAs that attenuate BCR signal transduction and consequently, dampen BCR-induced EBV reactivation, demonstrating that a subset of the viral miRNAs actively suppress lytic replication (31).…”

“…Additionally, studies in which Dicer was inhibited reported reduced levels of IE gene expression during EBV reactivation, providing evidence that components of the miRNA biogenesis machinery are necessary for aspects of lytic replication (33, 34). We previously reported that disruption of cellular miR-17 in EBV-positive BL cells augments IE gene expression (31), and other groups have shown that miR-200 family members expressed in epithelial cells have essential roles in the EBV latent to lytic switch (22, 23). In this study, we investigated how EBV exploits BCR-responsive cellular miRNAs to navigate entry into the lytic phase.…”