Interferon induced circRNAs escape herpesvirus host shutoff and suppress lytic infection.

To globally profile circRNAs, we employ RNA-Sequencing paired with chimeric junction analysis for alpha-, beta-, and gamma-herpesvirus infection. We find circRNAs are, as a population, resistant to host shutoff. We validate this observation using ectopic expression assays of human and murine herpesvirus endoribonucleases. During lytic infection, four circRNAs are commonly induced across all subfamilies of human herpesviruses, suggesting a shared mechanism of regulation. We test one such mechanism, namely how interferon-stimulation influences circRNA expression. 67 circRNAs are upregulated by either interferon-ß or -γ treatment, with half of these also upregulated during lytic infection. Using gain and loss of function studies we find an interferon-stimulated circRNA, circRELL1, inhibits lytic Herpes Simplex Virus-1 infection. We previously reported circRELL1 inhibits lytic Kaposi sarcoma-associated herpesvirus infection, suggesting a pan-herpesvirus antiviral activity. We propose a two-pronged model in which interferon-stimulated genes may encode both mRNA and circRNA with antiviral activity. This is critical in cases of host shutoff, such as alpha- and gamma-herpesvirus infection, where the mRNA products are degraded but circRNAs escape.

A virus-induced circular RNA maintains latent infection of Kaposi's sarcoma herpesvirus.

Non-coding RNAs (ncRNAs) play important roles in host-pathogen interactions; oncogenic viruses like Kaposi's sarcoma herpesvirus (KSHV) employ ncRNAs to establish a latent reservoir and persist for the life of the host. We previously reported that KSHV infection alters a novel class of RNA, circular RNAs (circRNAs). CircRNAs are alternative splicing isoforms and regulate gene expression, but their importance in infection is largely unknown. Here, we showed that a human circRNA, hsa_circ_0001400, is induced by various pathogenic viruses, namely KSHV, Epstein-Barr virus, and human cytomegalovirus. The induction of circRNAs including circ_0001400 by KSHV is co-transcriptionally regulated, likely at splicing. Consistently, screening for circ_0001400-interacting proteins identified a splicing factor, PNISR. Functional studies using infected primary endothelial cells revealed that circ_0001400 inhibits KSHV lytic transcription and virus production. Simultaneously, the circRNA promoted cell cycle, inhibited apoptosis, and induced immune genes. RNA-pull down assays identified transcripts interacting with circ_0001400, including TTI1, which is a component of the pro-growth mTOR complexes. We thus identified a circRNA that is pro-growth and anti-lytic replication. These results support a model in which KSHV induces circ_0001400 expression to maintain latency. Since circ_0001400 is induced by multiple viruses, this novel viral strategy may be widely employed by other viruses.

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

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.

Viral non-coding RNAs: Stealth strategies in the tug-of-war between humans and herpesviruses.

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.

Transcriptional landscape of Kaposi sarcoma tumors identifies unique immunologic signatures and key determinants of angiogenesis.

BACKGROUND: Kaposi sarcoma (KS) is a multicentric tumor caused by Kaposi sarcoma herpesvirus (KSHV) that leads to morbidity and mortality among people with HIV worldwide. KS commonly involves the skin but can occur in the gastrointestinal tract (GI) in severe cases. METHODS: RNA sequencing was used to compare the cellular and KSHV gene expression signatures of skin and GI KS lesions in 44 paired samples from 19 participants with KS alone or with concurrent KSHV-associated diseases. Analyses of KSHV expression from KS lesions identified transcriptionally active areas of the viral genome. RESULTS: The transcript of an essential viral lytic gene, ORF75, was detected in 91% of KS lesions. Analyses of host genes identified 370 differentially expressed genes (DEGs) unique to skin KS and 58 DEGs unique to GI KS lesions as compared to normal tissue. Interleukin (IL)-6 and IL-10 gene expression were higher in skin lesions as compared to normal skin but not in GI KS lesions. Twenty-six cellular genes were differentially expressed in both skin and GI KS tissues: these included Fms-related tyrosine kinase 4 (FLT4), encoding an angiogenic receptor, and Stanniocalcin 1 (STC1), a secreted glycoprotein. FLT4 and STC1 were further investigated in functional studies using primary lymphatic endothelial cells (LECs). In these models, KSHV infection of LECs led to increased tubule formation that was impaired upon knock-down of STC1 or FLT4. CONCLUSIONS: This study of transcriptional profiling of KS tissue provides novel insights into the characteristics and pathogenesis of this unique virus-driven neoplasm.

Identifying and characterizing virus-encoded circular RNAs.

Circular forms of RNA were first discovered in plant viroids and later found in a variety of animal viruses. These circular RNAs lack free 5' and 3' ends, granting protection from exonucleases. This review is focused on the methods that are used to investigate virus-encoded circular RNAs. Using DNA viruses that are prevalent among human as examples, we begin with features of circular RNAs and the unique methods to enrich for circular RNAs. Next, we discuss the computational methods for RNA-sequencing analysis to discover new virus-encoded circular RNAs. Many strategies are similar to analyzing cellular RNAs, but some unique aspects of virus-encoded circular RNAs that are likely due to highly packed viral genomes and non-canonical use of splicing machinery, are described herein. We illustrate the various methods of validating expression of specific virus-encoded circular RNAs. Finally, we discuss novel methods to study functions of circular RNAs and the current technical challenges that remain for investigating virus-encoded circular RNAs.

Discovery of Kaposi's sarcoma herpesvirus-encoded circular RNAs and a human antiviral circular RNA.

Noncoding RNAs have substantial effects in host-virus interactions. Circular RNAs (circRNAs) are novel single-stranded noncoding RNAs which can decoy other RNAs or RNA-binding proteins to inhibit their functions. The role of circRNAs is largely unknown in the context of Kaposi's sarcoma herpesvirus (KSHV). We hypothesized that circRNAs influence viral infection by inhibiting host and/or viral factors. Transcriptome analysis of KSHV-infected primary endothelial cells and a B cell line identified human circRNAs that are differentially regulated upon infection. We confirmed the expression changes with divergent PCR primers and RNase R treatment of specific circRNAs. Ectopic expression of hsa_circ_0001400, a circRNA induced by infection, suppressed expression of key viral latent gene LANA and lytic gene RTA in KSHV de novo infections. Since human herpesviruses express noncoding RNAs like microRNAs, we searched for viral circRNAs encoded in the KSHV genome. We performed circRNA-Seq analysis with RNase R-treated, circRNA-enriched RNA from KSHV-infected cells. We identified multiple circRNAs encoded by the KSHV genome that are expressed in KSHV-infected endothelial cells and primary effusion lymphoma (PEL) cells. The KSHV circRNAs are located within ORFs of viral lytic genes, are up-regulated upon the induction of the lytic cycle, and alter cell growth. Viral circRNAs were also detected in lymph nodes from patients of KSHV-driven diseases such as PEL, Kaposi's sarcoma, and multicentric Castleman's disease. We revealed new host-virus interactions of circRNAs: human antiviral circRNAs are activated in response to KSHV infection, and viral circRNA expression is induced in the lytic phase of infection.

Spatiotemporally Skewed Activation of Programmed Cell Death Receptor 1-Positive T Cells after Epstein-Barr Virus Infection and Tumor Development in Long-Term Fully Humanized Mice.

Humanized mice developing functional human T cells endogenously and capable of recognizing cognate human leukocyte antigen-matched tumors are emerging as relevant models for studying human immuno-oncology in vivo. Herein, mice transplanted with human CD34+ stem cells and bearing endogenously developed human T cells for >15 weeks were infected with an oncogenic recombinant Epstein-Barr virus (EBV), encoding enhanced firefly luciferase and green fluorescent protein. EBV-firefly luciferase was detectable 1 week after infection by noninvasive optical imaging in the spleen, from where it spread rapidly and systemically. EBV infection resulted into a pronounced immunologic skewing regarding the expansion of CD8+ T cells in the blood outnumbering the CD4+ T and CD19+ B cells. Furthermore, within 10 weeks of infections, mice developing EBV-induced tumors had significantly higher absolute numbers of CD8+ T cells in lymphatic tissues than mice controlling tumor development. Tumor outgrowth was paralleled by an up-regulation of the programmed cell death receptor 1 on CD8+ and CD4+ T cells, indicative for T-cell dysfunction. Histopathological examinations and in situ hybridizations for EBV in tumors, spleen, liver, and kidney revealed foci of EBV-infected cells in perivascular regions in close association with programmed cell death receptor 1-positive infiltrating lymphocytes. The strong spatiotemporal correlation between tumor development and the T-cell dysfunctional status seen in this viral oncogenesis humanized model replicates observations obtained in the clinical setting.

Epstein-Barr virus microRNAs reduce immune surveillance by virus-specific CD8+ T cells.

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.

MicroRNAs of Epstein-Barr Virus Control Innate and Adaptive Antiviral Immunity.

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.

A potent 2'-O-methylated RNA-based microRNA inhibitor with unique secondary structures.

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'-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.

Analysis of tumor infiltrating immune cells in Kaposi sarcoma lesions discovers shifts in macrophage populations.

Limited information exists about the types of immune cells present in Kaposi sarcoma (KS) lesions, especially in KS in the gastrointestinal tract. Using previously reported RNA-sequencing results from Kaposi sarcoma lesions in skin and gastrointestinal tract with normal matched tissues from the same patients at the same time, we investigated changes in lymphocytes in these tissues. We employed a computational method that determines changes in cell type distributions using KS lesion transcriptome data compared to a reference set of RNA expression patterns of purified immune cells. Since secreted cytokines and chemokines from KSHV-infected cells may influence the microenvironment of Kaposi sarcoma lesions, we performed cytokine profiling of conditioned media from KSHV-infected primary human dermal lymphatic endothelial cells. We also measured how this conditioned media altered the differentiation of macrophages in cell culture assays. These results suggested that factors in conditioned media from KSHV-infected endothelial cells promoted differentiation of a promonocytic cell line to proinflammatory macrophages.

Mapping herpesvirus-driven impacts on the cellular milieu and transcriptional profile of Kaposi sarcoma in patient-derived mouse models.

Kaposi sarcoma (KS) is defined by aberrant angiogenesis driven by Kaposi sarcoma herpesvirus (KSHV)-infected spindle cells with endothelial characteristics. KS research is hindered by rapid loss of KSHV infection upon explant culture of tumor cells. Here, we establish patient-derived KS xenografts (PDXs) upon orthotopic implantation of cutaneous KS biopsies in immunodeficient mice. KS tumors were maintained in 27/28 PDX until experimental endpoint, up to 272 days in the first passage of recipient mice. KSHV latency associated nuclear antigen (LANA)+ endothelial cell density increased by a mean 4.3-fold in 14/15 PDX analyzed by IHC at passage 1 compared to respective input biopsies, regardless of implantation variables and clinical features of patients. The Ki-67 proliferation marker colocalized with LANA more frequently in PDXs. Spatial transcriptome analysis revealed increased expression of viral transcripts from latent and lytic gene classes in the PDX. The expanded KSHV+ regions of the PDX maintained signature gene expression of KS tumors, with enrichment in pathways associated with angiogenesis and endothelium development. Cells with characteristics of tumor-associated fibroblasts derived from PDX were propagated for 15 passages. These fibroblast-like cells were permissive for de novo KSHV infection, and one lineage produced CXCL12, a cancer-promoting chemokine. Spatial analysis revealed that fibroblasts are a likely source of CXCL12 signaling to CXCR4 that was upregulated in KS regions. The reproducible expansion of KSHV-infected endothelial cells in PDX from multiple donors and recapitulation of a KS tumor gene signature supports the application of patient-derived KS mouse models for studies of pathogenesis and novel therapies.

Multiple microRNAs induced by Cdx1 suppress Cdx2 in human colorectal tumour 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.

Interferon induced circRNAs escape herpesvirus host shutoff and suppress lytic infection.

A first line of defense during infection is expression of interferon (IFN)-stimulated gene products which suppress viral lytic infection. To combat this, herpesviruses express endoribonucleases to deplete host RNAs. Here we demonstrate that IFN-induced circular RNAs (circRNAs) can escape viral-mediated degradation. We performed comparative circRNA expression profiling for representative alpha- (Herpes simplex virus-1, HSV-1), beta- (human cytomegalovirus, HCMV), and gamma-herpesviruses (Kaposi sarcoma herpesvirus, KSHV; murine gamma-herpesvirus 68, MHV68). Strikingly, we found that circRNAs are, as a population, resistant to host shutoff. This observation was confirmed by ectopic expression assays of human and murine herpesvirus endoribonucleases. During primary lytic infection, ten circRNAs were commonly regulated across all subfamilies of human herpesviruses, suggesting a common mechanism of regulation. We tested one such mechanism, namely how interferon-stimulation influences circRNA expression. 67 circRNAs were upregulated by either IFN-ß or -γ treatment, with half of these also upregulated during lytic infection. Using gain and loss of function studies we found an interferon-stimulated circRNA, circRELL1, inhibited lytic HSV-1 infection. We have previously reported circRELL1 inhibits lytic KSHV infection, suggesting a pan-herpesvirus antiviral activity. We propose a two-pronged model in which interferon-stimulated genes may encode both mRNA and circRNA with antiviral activity. This is critical in cases of host shutoff, such as alpha- and gamma-herpesvirus infection, where the mRNA products are degraded but circRNAs escape.

Strategies of Epstein-Barr virus to evade innate antiviral immunity of its human host.

Epstein-Barr virus (EBV) is a double-stranded DNA virus of the Herpesviridae family. This virus preferentially infects human primary B cells and persists in the human B cell compartment for a lifetime. Latent EBV infection can lead to the development of different types of lymphomas as well as carcinomas such as nasopharyngeal and gastric carcinoma in immunocompetent and immunocompromised patients. The early phase of viral infection is crucial for EBV to establish latency, but different viral components are sensed by cellular sensors called pattern recognition receptors (PRRs) as the first line of host defense. The efficacy of innate immunity, in particular the interferon-mediated response, is critical to control viral infection initially and to trigger a broad spectrum of specific adaptive immune responses against EBV later. Despite these restrictions, the virus has developed various strategies to evade the immune reaction of its host and to establish its lifelong latency. In its different phases of infection, EBV expresses up to 44 different viral miRNAs. Some act as viral immunoevasins because they have been shown to counteract innate as well as adaptive immune responses. Similarly, certain virally encoded proteins also control antiviral immunity. In this review, we discuss how the virus governs innate immune responses of its host and exploits them to its advantage.

25-Hydroxycholesterol Inhibits Kaposi's Sarcoma Herpesvirus and Epstein-Barr Virus Infections and Activates Inflammatory Cytokine Responses.

Oncogenic gammaherpesviruses express viral products during latent and lytic infection that block the innate immune response. Previously, we found that Kaposi's sarcoma herpesvirus (KSHV/human herpesvirus-8) viral microRNAs (miRNAs) downregulate cholesterol biogenesis, and we hypothesized that this prevents the production of 25-hydroxycholesterol (25HC), a cholesterol derivative. 25HC blocks KSHV de novo infection of primary endothelial cells at a postentry step and decreases viral gene expression of LANA (latency-associated nuclear antigen) and RTA. Herein we expanded on this observation by determining transcriptomic changes associated with 25HC treatment of primary endothelial cells using RNA sequencing (RNA-Seq). We found that 25HC treatment inhibited KSHV gene expression and induced interferon-stimulated genes (ISGs) and several inflammatory cytokines (interleukin 8 [IL-8], IL-1α). Some 25HC-induced genes were partially responsible for the broadly antiviral effect of 25HC against several viruses. Additionally, we found that 25HC inhibited infection of primary B cells by a related oncogenic virus, Epstein-Barr virus (EBV/human herpesvirus-4) by suppressing key viral genes such as LMP-1 and inducing apoptosis. RNA-Seq analysis revealed that IL-1 and IL-8 pathways were induced by 25HC in both primary endothelial cells and B cells. We also found that the gene encoding cholesterol 25-hydroxylase (CH25H), which converts cholesterol to 25HC, can be induced by type I interferon (IFN) in human B cell-enriched peripheral blood mononuclear cells (PBMCs). We propose a model wherein viral miRNAs target the cholesterol pathway to prevent 25HC production and subsequent induction of antiviral ISGs. Together, these results answer some important questions about a widely acting antiviral (25HC), with implications for multiple viral and bacterial infections. IMPORTANCE A cholesterol derivative, 25-hydroxycholesterol (25HC), has been demonstrated to inhibit infections from widely different bacteria and viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, its mechanism of activity is still not fully understood. In this work, we look at gene expression changes in the host and virus after 25HC treatment to find clues about its antiviral activity. We likewise demonstrate that 25HC is also antiviral against EBV, a common cancer-causing virus. We compared our results with previous data from antiviral screening assays and found the same pathways resulting in antiviral activity. Together, these results bring us closer to understanding how a modified form of cholesterol works against several viruses.

Transcriptomic Profiling Predicts Multiple Pathways and Molecules Associated With the Metastatic Phenotype of Oral Cancer Cells.

BACKGROUND/AIM: Metastasis to cervical lymph nodes of oral squamous cell carcinoma (OSCC) leads to a poor prognosis. The present study aimed at investigating the pathways and molecules associated with OSCC metastasis. MATERIALS AND METHODS: The transcriptome between HSC-3 cells and their highly metastatic subline, HSC-3-M3 cells, was examined using gene expression microarray. Gene enrichment analyses and Ingenuity Pathway Analysis were performed. Kaplan-Meier plot analysis using a publicly available dataset was conducted to assess whether candidate molecules are prognosticators. RESULTS: A total of 1,018 genes were differentially expressed, and the inflammatory pathway and NF-kB were predicted to be activated in HSC-3-M3 cells. CSF2 was suggested to be an indicator of poor prognosis in head and neck cancers. CONCLUSION: Inflammation and NF-kB may be involved in the metastasis of OSCC, and CSF2 is a promising diagnostic and therapeutic molecule. Moreover, HSC-3-M3 cells are a useful cell line model for studying OSCC progression.

Characterizing Expression and Regulation of Gamma-Herpesviral Circular RNAs.

Multiple herpesviruses have been recently found to encode viral circular RNAs. Like cellular circular RNAs, these RNAs lack poly-A tails and their 5' and 3' ends have been joined, which confers protection from RNA exonucleases. We examined the expression patterns of circular RNAs from Kaposi's sarcoma herpesvirus (KSHV) in various environments. We performed deep sequencing of circRNA-enriched total RNA from a KSHV-positive patient lymph node for comparison with previous circRNA-Seq results. We found that circvIRF4 is highly expressed in the KSHV-positive patient sample relative to both B cell lines and de novo infected primary vascular and lymphatic endothelial cells (LECs). Overall, this patient sample showed a viral circRNA expression pattern more similar to the pattern from B cell lines, but we also discovered new back-spliced junctions and additional viral circular RNAs in this patient sample. We validated some of these back-spliced junctions as circular RNAs with standard assays. Differential expression patterns of circular RNAs in different cell types led us to investigate what cellular factors might be influencing the ratio of viral linear mRNAs to circular RNAs. We found that repression of certain RNA-binding proteins shifted the balance between viral linear mRNAs and circular RNAs. Taken together, examining viral circular RNA expression patterns may become useful tools for discovering their functions, the regulators of their expression, and determining the stage and cell types of infection in humans.

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

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/ß 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.