Multiple Viral microRNAs Regulate Interferon Release and Signaling Early during Infection with Epstein-Barr Virus

Abstract

Epstein-Barr virus (EBV), a human herpesvirus, 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 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 prelatent phase of infection. The presence of epigenetically naive, 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 prelatent phase of infection and dampen the acute release of IFN-α in pDCs upon their encounter with EBV. IMPORTANCE Acute 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 that induce the 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 of Epstein-Barr virus (EBV) control over a dozen cellular genes that contribute to the antiviral 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.

MicroRNAs are minor constituents of extracellular vesicles that are rarely delivered to target cells

“…An incubation period longer than 4 hours did not lead to a marked signal increase (S4B Fig) . The EV field is still developing and standardized purification and analytical methods do not yet exist [53], but the ß-lactamase fusion assay has the potential to become a routine method for quantitating the uptake of EVs by acceptor cells in an unequivocal, background-free and quantitative manner. Recently, we successfully adapted this assay to characterize the fusion of EBV particles with different primary human immune cells [62] underlining the flexibility of our method.…”

Reevaluating the Impact of Epstein-Barr Virus Noncoding RNAs on the Interferon Response

“…In this context, the miRNAs and EBERs can be viewed as EBV-encoded virulence factors. A recent publication by Bouvet et al ( 8 ) reinvestigated the biological impact of encoded miRNAs and EBERs on the host antiviral response. With the addition of sequences found in clinical isolates of the virus that are lacking in laboratory bacterial artificial chromosome (BAC) strains, this study was able to generate a more-complete clinically relevant strain of the virus which was used to interrogate the requirement of the EBV miRNAs and EBERs.…”

“…With the addition of sequences found in clinical isolates of the virus that are lacking in laboratory bacterial artificial chromosome (BAC) strains, this study was able to generate a more-complete clinically relevant strain of the virus which was used to interrogate the requirement of the EBV miRNAs and EBERs. Building off this construct, the authors generated an array of highly engineered viral mutants lacking various ncRNAs or viral open reading frames (ORFs), all of which were previously reported to impact the interferon response upon infection ( 8 ). While generally there would be concerns that the simultaneous deletion of multiple viral factors might result in confounding results, the use of this physiologically relevant reagent allowed the authors to interrogate the requirement of these viral components in antiviral responses.…”

“…The surprising findings from infection of the primary cells revealed that the principal viral regulators of diminished cytokine production were the EBV miRNAs, whereas the EBERs and the viral protein LF2, previously characterized as a type I interferon antagonist ( 9 ), were largely nonessential for inhibiting this antiviral response. Further, by using both in silico prediction analysis and an unbiased miRNA target detection methodology, argonaute-photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (AGO PAR-CLIP), the authors not only identified multiple, key interferon-mediating factors as direct targets of the viral encoded miRNAs but found that ISG-driven transcription is negatively impacted in the presence of the miRNAs ( 8 ). Underscoring the effectiveness of undermining the interferon response, EBV employs redundancy in targeting interferon pathways by inhibiting several host factors which function at distinct stages of the same pathway.…”

“…As described above, there are two axes involved in the IFN-1 response: (i) pathogen detection inducing IFN production and (ii) the downstream signaling induced by IFN binding to its cognate receptor. Bouvet et al ( 8 ) found that EBV effectively inhibits both arms of the IFN response by targeting multiple factors in both pathogen recognition and IFN production, as well as mediators of the signal transduction cascade, whereas EBERs and LF2 had negligible effects on either arm in primary cells. This is a surprising finding, as previous studies have implicated the EBERs and LF2 as potent regulators of the interferon response ( 9 , 10 ).…”