B Cell Receptor-Responsive miR-141 Enhances Epstein-Barr Virus Lytic Cycle via FOXO3 Inhibition

Abstract

Antigen recognition by the B cell receptor (BCR) is a physiological trigger for reactivation of Epstein-Barr virus (EBV) and can be recapitulated in vitro by cross-linking of surface immunoglobulins. Previously, we identified a subset of EBV microRNAs (miRNAs) that attenuate BCR signal transduction and subsequently dampen lytic reactivation in B cells. The roles of host miRNAs in the EBV lytic cycle are not completely understood. Here, we profiled the small RNAs in reactivated Burkitt lymphoma cells and identified several miRNAs, such as miR-141, that are induced upon BCR cross-linking. Notably, EBV encodes a viral miRNA, miR-BART9, with sequence homology to miR-141. To better understand the functions of these two miRNAs, we examined their molecular targets and experimentally validated multiple candidates commonly regulated by both miRNAs. Targets included B cell transcription factors and known regulators of EBV immediate-early genes, leading us to hypothesize that these miRNAs modulate kinetics of the lytic cascade in B cells. Through functional assays, we identified roles for miR-141 and EBV miR-BART9 and one specific target, FOXO3, in progression of the lytic cycle. Our data support a model whereby EBV exploits BCR-responsive miR-141 and further mimics activity of this miRNA family via a viral miRNA to promote productive lytic replication. IMPORTANCE EBV is a human pathogen associated with several malignancies. A key aspect of lifelong virus persistence is the ability to switch between latent and lytic replication modes. The mechanisms governing latency, reactivation, and progression of the lytic cycle are only partly understood. This study reveals that specific miRNAs can act to support the EBV lytic phase following BCR-mediated reactivation triggers. Furthermore, this study identifies a role for FOXO3, commonly suppressed by both host and viral miRNAs, in modulating progression of the EBV lytic cycle.

MicroRNA-focused CRISPR/Cas9 Screen Identifies miR-142 as a Key Regulator of Epstein-Barr Virus Reactivation

“…1A). An LX-miR guide RNA (gRNA) library consisting of 8,382 individual gRNA constructs that were optimized to target 1,594 annotated human miRNA genes (∼85% of human miRNA genes with ∼4 gRNAs per miRNA) (32) was introduced in MutuI BL cells stably expressing a tet-inducible Cas9 (MutuI-iCas9) (26). This library also contains ∼1000 non-targeting control gRNAs.…”

“…In B cells, another miR-200 family member, miR-141, targets FOXO3 to support progression of the EBV lytic cycle when cells are stimulated to reactivate (26). Despite these few examples, the functions for most cellular miRNAs in modulating EBV infection, reactivation, and lytic cycle progression remain to be elucidated.…”

Epstein-Barr Virus miR-BARTs 7 and 9 modulate viral cycle, cell proliferation, and proteomic profiles in Burkitt lymphoma

“…EBV BART9 has been documented to modulate the EBV lytic cycle by transcriptionally inhibiting multiple elements within the BCR and FOXO3 signaling pathway. Furthermore, it's worth noting the considerable sequence homology shared between EBV BART9 and the human miRNA miR-141 [ 28 , 29 ], a crucial regulator of various signaling pathways implicated in the development and progression of human cancers [ 30 ]. In summary, these results support the hypothesis that the expression of EBV miRs BART-7 and 9 regulate cell proliferation and apoptosis and maintain viral latency in malignant B-cells derived from BL.…”

Virus-Induced Tumorigenesis and IFN System

“…It is well known that FOXO3 protein dampens type I IFNs response through the suppression of IRF7. EBV regulates the FOXO3 activities through the expression of miR-BART9 and the upregulation on human miR-141 thus targeting ZCCHC3, a cosensor for cGAS protein to manage the shift from latent to lytic life cycle [113] (Figure 5).…”