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Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells’ transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we defined multiple properties of EVs and analyzed their capacity to deliver packaged miRNAs into target cells to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but EVs did not fuse detectably with cellular membranes to deliver their cargo. We engineered EVs to be fusogenic and document their capacity to deliver functional messenger RNAs. Engineered fusogenic EVs, however, did not detectably alter the functionality of cells exposed to miRNA-carrying EVs. These results suggest that EV-borne miRNAs do not act as effectors of cell-to-cell communication.

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)., 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.

Oncogenic DNA viruses establish lifelong infections in humans, and they cause cancers, often in immunocompromised patients, despite anti-viral immune surveillance targeted against viral antigens. High-throughput sequencing techniques allowed the field to identify novel viral non-coding RNAs (ncRNAs). ncRNAs are ideal factors for DNA viruses to exploit; they are non-immunogenic to T cells, thus viral ncRNAs can manipulate host cells without evoking adaptive immune responses. Viral ncRNAs may still trigger the host innate immune response, but many viruses encode decoys/inhibitors to counter-act and evade recognition. In addition, ncRNAs can be secreted to the extracellular space and influence adjacent cells to create a pro-viral microenvironment. In this review, we present recent progress in understanding interactions between oncoviruses and ncRNAs including small and long ncRNAs, microRNAs, and recently identified viral circular RNAs. In addition, potential clinical applications for ncRNA will be discussed. Extracellular ncRNAs are suggested to be diagnostic and prognostic biomarkers and, with the realization of the importance of viral ncRNAs in tumorigenesis, approaches to target critical viral ncRNAs are emerging. Further understanding of viral utilization of ncRNAs will advance anti-viral therapeutics beyond conventional medication and vaccination.

EBV reduces the activation of cytotoxic CD4+ effector T cells by inducing a state of reduced immunogenicity in infected B cells. EBV-derived miRNAs suppress release of proinflammatory cytokines, interfere with peptide processing and presentation on HLA class II, repress differentiation of naive CD4+ T cells to Th1 cells, and ultimately avoid killing of infected B cells., Epstein-Barr virus (EBV) is a tumor virus that establishes lifelong infection in most of humanity, despite eliciting strong and stable virus-specific immune responses. EBV encodes at least 44 miRNAs, most of them with unknown function. Here, we show that multiple EBV miRNAs modulate immune recognition of recently infected primary B cells, EBV's natural target cells. EBV miRNAs collectively and specifically suppress release of proinflammatory cytokines such as IL-12, repress differentiation of naive CD4+ T cells to Th1 cells, interfere with peptide processing and presentation on HLA class II, and thus reduce activation of cytotoxic EBV-specific CD4+ effector T cells and killing of infected B cells. Our findings identify a previously unknown viral strategy of immune evasion. By rapidly expressing multiple miRNAs, which are themselves nonimmunogenic, EBV counteracts recognition by CD4+ T cells and establishes a program of reduced immunogenicity in recently infected B cells, allowing the virus to express viral proteins required for establishment of life-long infection.

Epstein-Barr virus (EBV) has established lifelong infection in more than 90% of humanity. While infection is usually controlled by the immune system, the human host fails to completely eliminate the pathogen. Several herpesviral proteins are known to act as immunoevasins, preventing or reducing recognition of EBV-infected cells. Only recently were microRNAs of EBV identified to reduce immune recognition further. This Gem summarizes what we know about immunomodulatory microRNAs of herpesviruses.

Epstein-Barr virus (EBV) has evolved exquisite controls over its host cells, human B lymphocytes, not only directing these cells during latency to proliferate and thereby expand the pool of infected cells, but also to survive and thereby persist for the lifetime of the infected individual. Although these activities ensure the virus is successful, they also make the virus oncogenic, particularly when infected people are immunosuppressed. Here we show, strikingly, that one set of EBV’s miRNAs both sustain BL (Burkitt’s lymphoma) cells in the absence of other viral oncogenes and promote the transformation of primary B lymphocytes. Burkitt’s Lymphoma cells were engineered to lose EBV and found to die by apoptosis and could be rescued by constitutively expressing viral miRNAs in them. Two of these EBV miRNAs were found to target Caspase 3 to inhibit apoptosis at physiological concentrations.

Infection with Epstein-Barr virus (EBV) affects most humans worldwide and persists life-long in the presence of robust virus-specific T-cell responses. In both immunocompromised and some immunocompetent people, EBV causes several cancers and lymphoproliferative diseases. EBV transforms B cells in vitro and encodes at least 44 microRNAs (miRNAs), most of which are expressed in EBV-transformed B cells, but their functions are largely unknown. Recently, we showed that EBV miRNAs inhibit CD4(+) T-cell responses to infected B cells by targeting IL-12, MHC class II, and lysosomal proteases. Here we investigated whether EBV miRNAs also counteract surveillance by CD8(+) T cells. We have found that EBV miRNAs strongly inhibit recognition and killing of infected B cells by EBV-specific CD8(+) T cells through multiple mechanisms. EBV miRNAs directly target the peptide transporter subunit TAP2 and reduce levels of the TAP1 subunit, MHC class I molecules, and EBNA1, a protein expressed in most forms of EBV latency and a target of EBV-specific CD8(+) T cells. Moreover, miRNA-mediated down-regulation of the cytokine IL-12 decreases the recognition of infected cells by EBV-specific CD8(+) T cells. Thus, EBV miRNAs use multiple, distinct pathways, allowing the virus to evade surveillance not only by CD4(+) but also by antiviral CD8(+) T cells.

The mammalian transcriptional factors, Cdx1 and Cdx2 (Cdx is caudal-type homeobox) are paralogues and critical for the cellular differentiation of intestinal or colorectal epithelia. It has been reported previously that in Cdx1 transgenic or knockout mice, endogenous Cdx2 levels are inversely correlated with Cdx1 levels. Recently, we found that exogenous Cdx1 expression can suppress Cdx2 in a human colorectal tumour cell line, SW480, although the underlying molecular mechanisms were unclear. In the present study, we show that several microRNAs induced by exogenous Cdx1 expression directly bind to the CDX2 mRNA 3′UTR (untranslated region) to destabilize these transcripts, finally leading to their degradation. Using microarray analysis, we found that several miRNAs that were computationally predicted to target CDX2 mRNAs are up-regulated by exogenous Cdx1 expression in SW480 cells. Among these molecules, we identified miR-9 , miR-16 and miR-22 as having the potential to suppress Cdx2 through the binding of the 3′UTR to its transcript. Importantly, simultaneous mutations of both the miR-9 - and miR-16 -binding sites in the CDX2 3′UTR were shown to be sufficient to block Cdx2 suppression. The results of the present study suggest a unique feature of miRNAs in which they contribute to homoeostasis by limiting the levels of transcription factors belonging to the same gene family. Abbreviations: CDX/Cdx, caudal-type homeobox; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miRNA, microRNA; RT, reverse transcription; UTR, untranslated region; VSV-G, vesicular stomatitis virus glycoprotein

The chromatin remodeling complex SWI/SNF is an important epigenetic regulator that includes one Brm or BRG1 molecule as catalytic subunit. Brm and BRG1 do not function identically, so this complex can regulate gene expression either positively or negatively, depending on the promoter to which it is recruited. Notably, Brm attenuation due to posttranscription suppression occurs often in human tumor cells, in which this event contributes to their oncogenic potential. Here, we report that the 3′-untranslated region of Brm mRNA has two sites that are efficiently targeted by the microRNAs miR-199a-5p and -3p, revealing a novel mechanism for modulation of Brm-type SWI/SNF activity. Computational mapping of the putative promoter region of miR-199a-2 (miPPR-199a-2) has defined it as the major contributing genetic locus for miR-199a-5p and-3p production in these tumor cell lines. We validated this predicted region by direct promoter analysis to confirm that Egr1 is a strong positive regulator of the miR-199a-2 gene. Importantly, we also showed that Egr1, miR-199a-5p, and miR-199a-3p are expressed at high levels in Brm-deficient tumor cell lines but only marginally in Brm-expressing tumor cells. Finally, we also obtained evidence that Brm negatively regulates Egr1. Together, our results reveal that miR-199a and Brm form a double-negative feedback loop through Egr1, leading to the generation of these two distinct cell types during carcinogenesis. This mechanism may offer a partial explanation for why miR-199a-5p and -3p have been reported to be either upregulated or downregulated in a variety of tumors. Cancer Res; 71(5); 1680–9. ©2010 AACR.

MicroRNAs (miRNAs) are involved in various biological processes and human diseases. The development of strong low-molecular weight inhibitors of specific miRNAs is thus expected to be useful in providing tools for basic research or in generating promising new therapeutic drugs. We have previously described the development of ‘Tough Decoy (TuD) RNA’ molecules, which achieve the long-term suppression of specific miRNA activity in mammalian cells when expressed from a lentivirus vector. In our current study, we describe new synthetic miRNA inhibitors, designated as S-TuD (Synthetic TuD), which are composed of two fully 2 0 -O-methylated RNA strands. Each of these strands includes a miRNA-binding site. Following the hybridization of paired strands, the resultant S-TuD forms a secondary structure with two stems, which resembles the corresponding TuD RNA molecule. By analyzing the effects of S-TuD against miR-21, miR-200c, miR-16 and miR-106b, we have elucidated the critical design features of S-TuD molecules that will provide optimum inhibitory effects following transfection into human cell lines. We further show that the inhibitory effects of a single transfection of S-TuD-miR200c are quite long-lasting (>7 days) and induce partial EMT, the full establishment of which requires 11 days when using a lentivirus vector that expresses TuD-miR200c continuously.