Date Published: January 7, 2019
Publisher: Public Library of Science
Author(s): Yan Chen, Devin Fachko, Nikita S. Ivanov, Camille M. Skinner, Rebecca L. Skalsky, Zhen Lin.
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.
Epstein Barr virus (EBV) is a human gamma-herpesvirus that infects >90% of adults worldwide. Primary infection is often asymptomatic or presents as infectious mononucleosis. In immunocompromised individuals, the virus is linked to post-transplant lymphoproliferative disease (PTLD) and hematological malignancies including Burkitt’s (BL) lymphoma, Hodgkin lymphoma, and AIDS-related lymphoma; cancers of epithelial origin such as gastric and nasopharyngeal carcinomas are also associated with EBV infection [1,2]. Following EBV transmission through the oral cavity and subsequent infection of naïve B cells, EBV co-opts multiple aspects of normal B cell activation that induces cell proliferation, initiates differentiation programs, and can drive infected B cells through germinal center (GC) reactions to establish latency in the memory B cell compartment [3–5]. Periodic virus reactivation can occur throughout life-long infection of the host and is thought to help maintain the pool of latently infected cells [5,6]. Like all herpesviruses, EBV has both latent and lytic replication phases, and key to the success of long-term persistence is the ability to navigate between these phases.
In this study, we evaluated the role of EBV miRNAs in BCR signaling, which has implications for the proliferation of latently infected B cells and importantly, for influencing virus reactivation from the latent state. Antigen stimulation of the BCR induces multiple intracellular signaling pathways, and latent viral proteins have been previously demonstrated to manipulate BCR signaling components and/or the pathways (i.e. NF-kappaB, AP1, PI3K, MAPK) triggered through BCR activation. As viral miRNAs are non-immunogenic viral gene products that are expressed throughout multiple stages of EBV infection, we hypothesized that the viral miRNAs would be prime candidates to coordinately modulate the signaling cascade initiated through the BCR.