Multiple viral microRNAs regulate interferon release and signaling early during infection with Epstein-Barr virus

Abstract

Epstein-Barr virus (EBV), a human herpes virus, encodes 44 microRNAs (miRNAs), which regulate many genes with various functions in EBV-infected cells. Multiple target genes of the EBV miRNAs have been identified, some of which play important roles in adaptive antiviral immune responses. Using EBV mutant derivatives, we identified additional roles of viral miRNAs in governing versatile type I interferon (IFN) responses upon infection of human primary mature B cells. We also found that Epstein-Barr virus-encoded small RNAs (EBERs) and LF2, viral genes with previously reported functions in inducing or regulating IFN-I pathways, had negligible or even contrary effects on secreted IFN-α in our model. Data mining and Ago PAR-CLIP experiments uncovered more than a dozen of previously uncharacterized, direct cellular targets of EBV miRNA associated with type I IFN pathways. We also identified indirect targets of EBV miRNAs in B cells, such as TRL7 and TLR9, in the pre-latent phase of infection. The presence of epigenetically naïve, non-CpG methylated viral DNA was essential to induce IFN-α secretion during EBV infection in a TLR9-dependent manner. In a newly established fusion assay, we verified that EBV virions enter a subset of plasmacytoid dendritic cells (pDCs) and determined that these infected pDCs are the primary producers of IFN-α in EBV-infected peripheral blood mononuclear cells. Our findings document that many EBV-encoded miRNAs regulate type I IFN response in newly EBV infected primary human B cells in the pre-latent phase of infection and dampen the acute release of IFN-α in pDCs upon their encounter with EBV.Author summaryAcute antiviral functions of all nucleated cells rely on type I interferon (IFN-I) pathways triggered upon viral infection. Host responses encompass the sensing of incoming viruses, the activation of specific transcription factors which induce transcription of IFN-I genes, the secretion of different IFN-I types and their recognition by the heterodimeric IFN-α/β receptor, the subsequent activation of JAK/STAT signaling pathways and, finally, the transcription of many IFN-stimulated genes (ISGs). In sum, these cellular functions establish a so-called antiviral state in infected and neighboring cells. To counteract these cellular defense mechanisms, viruses have evolved diverse strategies and encode gene products that target antiviral responses. Among such immune evasive factors are viral microRNAs (miRNAs) that can interfere with host gene expression. We discovered that multiple miRNAs encoded by Epstein-Barr virus (EBV) control over a dozen cellular genes that contribute to the anti-viral states of immune cells, specifically B cells and plasmacytoid dendritic cells (pDCs). We identified the viral DNA genome as the activator of IFN-α and question the role of abundant EBV EBERs, that, contrary to previous reports, do not have an apparent inducing function in the IFN-I pathway early after infection.

Discovery of frog virus 3 microRNAs and their roles in evasion of host antiviral responses

“…v-miRNAs are emerging post-transcriptional regulators of host immune responses and have been ascribed immunoevasive functions [reviewed in (Cullen, 2013)]. For example, v-miRNAs produced by other dsDNA viruses (EBV, hCMV, KSHV) have been found to target host antiviral genes resulting in repression of proinflammatory cytokine production and release (Abend et al, 2012; Abend et al, 2010; Bouvet et al, 2021; Hancock et al, 2017; Hook et al, 2014; Landais et al, 2015; Lau et al, 2016; Lei et al, 2010). Accordingly, target prediction and pathway analyses revealed that FV3 v-miRNAs are predicted to target host genes involved in several antiviral pathways such as the cGAS-STING, RIG-I/MDA5, TLR3, TLR4, TLR9, and type I IFN signaling pathways, as well as the downstream products of signaling pathways triggered by virus sensing by these PRRs (NF-κB-induced genes and ISGs), and the impacts of these potential interactions are discussed below.…”

“…This suggests that FV3 has evolved the ability to simultaneously impact at least five different antiviral signaling pathways that defend against viruses at the cell membrane, in the cytoplasm, and in the endosome. As impairment of type I IFN signaling has been observed in other viruses such as EBV, hCMV, and KSHV (Abend et al, 2012; Bouvet et al, 2021; Hooykaas et al, 2017; Huang and Lin, 2014; Huang et al, 2015), it will be important to functionally characterize fv3-miR-8-3p, fv3-miR-7-5p, fv3-miR-27-5p, and fv3-miR-28-5p which target mediators of type I IFN signaling. It is also interesting to note that FV3 v-miRNAs appear to mostly target the RIG-I/MDA5 cytoplasmic sensing pathway by specifically targeting genes involved in ubiquitination ( trim25, usp4, usp38 , and trim11 ), suggesting that FV3 may largely regulate this pathway by modulating ubiquitination.…”