Dai Iwakiri

EB virus‐encoded RNAs are recognized by RIG‐I and activate signaling to induce type I IFN

Epstein–Barr virus (EBV)‐encoded small RNAs (EBERs) are nonpolyadenylated, untranslated RNAs, exist most abundantly in latently EBV‐infected cells, and are expected to show secondary structures with many short stem–loops. Retinoic acid‐inducible gene I (RIG‐I) is a cytosolic protein that detects viral double‐stranded RNA (dsRNA) inside the cell and initiates signaling pathways leading to the induction of protective cellular genes, including type I interferons (IFNs). We investigated whether EBERs were recognized by RIG‐I as dsRNA. Transfection of RIG‐I plasmid induced IFNs and IFN‐stimulated genes (ISGs) in EBV‐positive Burkitts lymphoma (BL) cells, but not in their EBV‐negative counterparts or EBER‐knockout EBV‐infected BL cells. Transfection of EBER plasmid or in vitro‐synthesized EBERs induced expression of type I IFNs and ISGs in RIG‐I‐expressing, EBV‐negative BL cells, but not in RIG‐I‐minus counterparts. EBERs activated RIG‐Is substrates, NF‐κB and IFN regulatory factor 3, which were necessary for type I IFN activation. It was also shown that EBERs co‐precipitated with RIG‐I. These results indicate that EBERs are recognized by RIG‐I and activate signaling to induce type I IFN in EBV‐infected cells.

Epstein-Barr virus (EBV)–encoded small RNA is released from EBV-infected cells and activates signaling from toll-like receptor 3

Epstein-Barr virus–encoded small RNA (EBER) is nonpolyadenylated, noncoding RNA that forms stem-loop structure by intermolecular base-pairing, giving rise to double-stranded RNA (dsRNA)–like molecules, and exists abundantly in EBV-infected cells. Here, we report that EBER induces signaling from the Toll-like receptor 3 (TLR3), which is a sensor of viral double-stranded RNA (dsRNA) and induces type I IFN and proinflammatory cytokines. A substantial amount of EBER, which was sufficient to induce signaling from TLR3, was released from EBV-infected cells, and the majority of the released EBER existed as a complex with a cellular EBER-binding protein La, suggesting that EBER was released from the cells by active secretion of La. Sera from patients with infectious mononucleosis (IM), chronic active EBV infection (CAEBV), and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), whose general symptoms are caused by proinflammatory cytokines contained EBER, and addition of RNA purified from the sera into culture medium induced signaling from TLR3 in EBV-transformed lymphocytes and peripheral mononuclear cells. Furthermore, DCs treated with EBER showed mature phenotype and antigen presentation capacity. These findings suggest that EBER, which is released from EBV-infected cells, is responsible for immune activation by EBV, inducing type I IFN and proinflammatory cytokines. EBER-induced activation of innate immunity would account for immunopathologic diseases caused by active EBV infection.

Editing of Epstein-Barr virus-encoded BART6 microRNAs controls their dicer targeting and consequently affects viral latency.

Certain primary transcripts of miRNA (pri-microRNAs) undergo RNA editing that converts adenosine to inosine. The Epstein-Barr virus (EBV) genome encodes multiple microRNA genes of its own. Here we report that primary transcripts of ebv-miR-BART6 (pri-miR-BART6) are edited in latently EBV-infected cells. Editing of wild-type pri-miR-BART6 RNAs dramatically reduced loading of miR-BART6-5p RNAs onto the microRNA-induced silencing complex. Editing of a mutation-containing pri-miR-BART6 found in Daudi Burkitt lymphoma and nasopharyngeal carcinoma C666-1 cell lines suppressed processing of miR-BART6 RNAs. Most importantly, miR-BART6-5p RNAs silence Dicer through multiple target sites located in the 3′-UTR of Dicer mRNA. The significance of miR-BART6 was further investigated in cells in various stages of latency. We found that miR-BART6-5p RNAs suppress the EBNA2 viral oncogene required for transition from immunologically less responsive type I and type II latency to the more immunoreactive type III latency as well as Zta and Rta viral proteins essential for lytic replication, revealing the regulatory function of miR-BART6 in EBV infection and latency. Mutation and A-to-I editing appear to be adaptive mechanisms that antagonize miR-BART6 activities.

Epstein-Barr virus-encoded small RNA induces IL-10 through RIG-I-mediated IRF-3 signaling.

Epstein–Barr virus-encoded small RNA (EBER) is nonpolyadenylated, noncoding RNA, forms stem-loop structure by intermolecular base-pairing giving rise to double-stranded RNA (dsRNA)-like molecule and exists abundantly in EBV-infected cells. EBER induces IL-10 and thus supports the growth of Burkitts lymphoma (BL) cells. In this study, the mechanism of IL-10 induction by EBER was analysed in the context of dsRNA signaling pathway. Knockdown of retinoic acid-inducible gene I (RIG-I) by small interfering RNA (siRNA), and expression of dominant-negative RIG-I downregulated IL-10 induction in EBER(+) EBV-infected and EBER plasmid-transfected BL cells. Transfection of EBER-expressing plasmid or in vitro synthesized EBER induced IL-10 in RIG-I-expressing cell clones, and activation of IL-10 promoter by EBER was blocked by dominant-negative RIG-I. Blocking of nuclear factor (NF)-κB by dominant-negative IκB-α plasmid did not block IL-10 expression, whereas knockdown of IRF-3 by siRNA resulted in downregulation of IL-10 in EBER(+) BL cells. NF-κB is reported to function downstream of RIG-I signaling pathway and is involved in the induction of proinflammatory cytokines. Our results indicate that EBER induces an anti-inflammatory cytokine IL-10 through RIG-I-mediated IRF-3 but not NF-κB signaling. These findings suggest a new mechanism of dsRNA signaling pathway that triggers the expression of IL-10, which acts as an autocrine growth factor in BL cells.

Epstein–Barr virus-encoded small RNA induces insulin-like growth factor 1 and supports growth of nasopharyngeal carcinoma-derived cell lines

To assess the role of insulin-like growth factor 1 (IGF-1) in the growth of nasopharyngeal carcinoma (NPC), three NPC-derived cell lines, C666-1, CNE1 and HONE1, were examined. C666-1 cells maintained NPC phenotype of Epstein–Barr virus (EBV) expression and were positive for IGF-1 secretion, and their growth was strikingly inhibited by treatment with an anti-IGF-1 antibody under low serum condition. On the other hand, CNE1 and HONE1 cells were EBV-negative and did not secrete IGF-1. Although they could not grow under low serum condition, addition of recombinant IGF-1 made them grow. EBV conversion of CNE1 and HONE1 cells reproduced NPC phenotype of EBV expression and accompanied IGF-1 expression. Although they could grow under low serum condition, their growth was strikingly inhibited by treatment with the anti-IGF-1 antibody. These results suggest that EBV infection induces IGF-1 in NPC cell lines, and that the secreted IGF-1 acts as an autocrine growth factor. These findings seem to be operative in vivo, as NPC biopsies consistently express IGF-1. Further studies demonstrated that increased IGF-1 expression reflected transcriptional activation, and EBV-encoded small RNA (EBER) was responsible for IGF-1 induction. EBER is invariably expressed in EBV-associated malignancies, including NPC. The present findings strongly suggest that EBER directly affects the pathogenesis of NPC.

Epstein-Barr virus nuclear protein EBNA3C is required for cell cycle progression and growth maintenance of lymphoblastoid cells.

Epstein–Barr virus (EBV) infection converts primary human B cells into continuously proliferating lymphoblastoid cell lines (LCLs). To examine the role of EBV nuclear antigen (EBNA) 3C in the proliferation of LCLs, we established LCLs infected with an EBV recombinant that expresses EBNA3C with a C-terminal fusion to a 4-hydroxytamoxifen (4HT)-dependent mutant estrogen receptor, E3C–HT. In the presence of 4HT, LCLs expressed the E3C–HT protein and grew like WT LCLs. When E3C–HT EBV-infected LCLs were transferred to medium without 4HT, E3C–HT protein slowly disappeared, and the LCLs gradually ceased growing. WT EBNA3C expression from an oriP plasmid transfected into E3C–HT LCLs protected the LCLs from growth arrest in medium without 4HT, whereas expression of EBNA3A or EBNA3B did not. The expression of other EBNA proteins and of LMP1, CD21, CD23, and c-myc was unaffected by EBNA3C inactivation. However, EBNA3C inactivation resulted in the accumulation of p16INK4A, a decrease in the hyperphosphorylated form of the retinoblastoma protein, and a decrease in the proportion of cells in S or G2/M phase. These results indicate that EBNA3C has an essential role in cell cycle progression and the growth maintenance of LCLs.

A novel animal model of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in humanized mice

EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a rare yet devastating disorder caused by EBV infection in humans. However, the mechanism of this disease has yet to be elucidated because of a lack of appropriate animal models. Here, we used a human CD34(+) cell-transplanted humanized mouse model and reproduced pathologic conditions resembling EBV-HLH in humans. By 10 weeks postinfection, two-thirds of the infected mice died after exhibiting high and persistent viremia, leukocytosis, IFN-γ cytokinenemia, normocytic anemia, and thrombocytopenia. EBV-infected mice also showed systemic organ infiltration by activated CD8(+) T cells and prominent hemophagocytosis in BM, spleen, and liver. Notably, the level of EBV load in plasma correlated directly with both the activation frequency of CD8(+) T cells and the level of IFN-γ in plasma. Moreover, high levels of EBV-encoded small RNA1 were detected in plasma of infected mice, reflecting what has been observed in patients. These findings suggest that our EBV infection model mirrors virologic, hematologic, and immunopathologic aspects of EBV-HLH. Furthermore, in contrast to CD8(+) T cells, we found a significant decrease of natural killer cells, myeloid dendritic cells, and plasmacytoid dendritic cells in the spleens of infected mice, suggesting that the collapse of balanced immunity associates with the progression of EBV-HLH pathogenesis.

Epstein-Barr Virus BZLF1 Gene, a Switch from Latency to Lytic Infection, Is Expressed as an Immediate-Early Gene after Primary Infection of B Lymphocytes

ABSTRACT We demonstrate here that the Epstein-Barr virus (EBV) BZLF1 gene, a switch from latent infection to lytic infection, is expressed as early as 1.5 h after EBV infection in Burkitts lymphoma-derived, EBV-negative Akata and Daudi cells and primary B lymphocytes. Since BZLF1 mRNA is expressed even when the cells are infected with EBV in the presence of anisomycin, an inhibitor of protein synthesis, its expression does not require prerequisite protein synthesis, indicating that BZLF1 is expressed as an immediate-early gene following primary EBV infection of B lymphocytes.

Role of EBERs in the pathogenesis of EBV infection.

Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) are noncoding RNAs that are expressed abundantly in latently EBV-infected cells. Previous studies demonstrated that EBERs (EBER1 and EBER2) play significant roles in various EBV-infected cancer cells. EBERs are responsible for malignant phenotypes of Burkitts lymphoma (BL) cells including resistance to apoptosis. In addition, EBERs induce the expression of interleukin (IL)-10 in BL cells, insulin-like growth factor (IGF)-1 in gastric carcinoma and nasopharyngeal carcinoma cells, IL-9 in T cells that act as an autocrine growth factor. It was also reported that EBERs play critical roles in the B cell growth transformation including IL-6 induction by EBER2. EBERs have been discovered to interact with cellular proteins that play a key role in antiviral innate immunity. They bind the protein kinase RNA-dependent (PKR) and inhibit its activation, leading to resistance to PKR-mediated apoptosis. Recently, it was demonstrated that EBERs bind RIG-I and activate its downstream signaling, which induces expression of type-I interferon (IFN)s. Furthermore, EBERs induce IL-10 through IRF3 but not NF-kappaB activation in BL cells, suggesting that modulation of innate immune signaling by EBERs contribute to EBV-mediated oncogenesis. Most recently, it was reported that EBERs are secreted from EBV-infected cells and are recognized by toll-like receptor (TLR)3, leading to induction of type-I IFNs and inflammatory cytokines, and subsequent immune activation. Furthermore, EBER1 could be detected in the sera of patients with active EBV infectious diseases, suggesting that activation of TLR3 signaling by EBER1 would be account for the pathogenesis of active EBV infectious diseases.

Symmetrical localization of extrachromosomally replicating viral genomes on sister chromatids

In eukaryotes, many latent viruses replicate as extrachromosomal molecules, called episomes, and efficiently segregate to daughter cells by noncovalently attaching to mitotic chromosomes. To understand the mechanism governing the processes, we analyzed the detailed subcellular localization of Epstein-Barr virus (EBV) genomes and a viral protein EBNA1, a bridging molecule between viral genomes and cellular chromatin. In the cells that were infected with a recombinant EBV expressing epitope-tagged EBNA1, EBNA1 localized to intranuclear punctate dots, which coincided with the localization of EBV genomes as revealed by fluorescence in situ hybridization (FISH). A significant number of EBNA1 dots were found to localize symmetrically on sister chromatids of mitotic chromosomes. Such symmetrical localization of EBNA1 dots was observed in prematurely condensed G2 chromosomes as well, correlating with the presence of closely spaced double dots of EBNA1 in G2-phase-enriched cells. The EBNA1 double dots were occasionally interconnected by the FISH signals of EBV episomes, exhibiting a dumbbell-like appearance. Thus, we propose that the partitioning of EBNA1 molecules onto sister chromatids during cellular DNA replication underlies the non-stochastic segregation of extrachromosomally replicating viral genomes.