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. 2009 Sep 28;206(10):2091-9.
doi: 10.1084/jem.20081761. Epub 2009 Aug 31.

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

Dai Iwakiri  1 , Li Zhou, Mrinal Samanta, Misako Matsumoto, Takashi Ebihara, Tsukasa Seya, Shosuke Imai, Mikiya Fujieda, Keisei Kawa, Kenzo Takada
Affiliations

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

Dai Iwakiri et al. J Exp Med. .

Abstract

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.

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Figures

Figure 1.
Figure 1.
EBER1 is released into the culture supernatants of EBV-infected Mutu+, Akata+ and LCL. (A) RT-PCR of EBER1. Cells (2 × 105 cells/ml) were cultured for the designated number of days. Total RNA was extracted from 1 ml culture supernatant and subjected to 25 cycles of RT-PCR to detect EBER1. Three or more independent experiments were performed. (B) Quantitative RT-PCR of EBER1 and EBER2. Cells (2 × 105) were cultured in 1 ml medium for 4 d, and total RNA was extracted from the culture supernatant and subjected to RT-RCR for the detection of EBER1 and EBER2. Error bars indicate the SD of duplicate wells. The data presented are representative of three independent experiments.
Figure 2.
Figure 2.
EBER1 activates signaling through TLR3. (A) Detection of TLR3 in three LCL clones and EBV-infected and uninfected NU-GC-3 cells. Total RNA (0.1 µg) was subjected to 30 cycles of RT-PCR to detect TLR3. RT-PCR for GAPDH was used as an internal control. Three or more independent experiments were performed for each assay. (B) Effect of in vitro–synthesized EBER1 on the expression of IFN-β. LCLs and EBV-positive and –negative NU-GC-3 cells were treated with 0.5 µg/ml in vitro–synthesized EBER1 or poly(I:C) and were cultured for 14 h. Total RNA (0.1 µg) was subjected to 30 cycles of RT-PCR to detect IFN-β. RT-PCR for GAPDH was used as an internal control. Three or more independent experiments were performed. (C) Dose response of the effect of in vitro–synthesized EBER1 on the expression of IFN-β. LCLs (4 × 105 cells/ml) were treated with 0.1–1,000 ng/ml EBER1 and cultured for 14 h. IFN-β in culture supernatants was quantified by ELISA. Error bars indicate the S.D. of duplicate wells. The data presented are representative of three independent experiments. (D) Efficiency of EBER1 and poly(I:C) to induce IFN-β expression in LCLs. LCLs (4 × 105 cells/ml) were treated with 1–1,000 ng/ml EBER1 and cultured for 14 h. IFN-β induction was analyzed by RT-PCR. Three or more independent experiments were performed for each assay. (E) Effect of an anti-TLR3 antibody on EBER1-induced IFN-β production. LCLs were preincubated with the anti-TLR3 antibody for 30 min at 37°C, before being treated with 0.5 µg/ml of EBER1 and incubated for 14 h. The culture was analyzed for IFN-β induction by RT-PCR (upper panel) and ELISA (lower panel). Error bars indicate the SD of duplicate wells. The data presented are representative of three independent experiments. (F) Effect of TLR3 knockdown on EBER1-induced IFN-β production. Negative control siRNA (nc) or TLR3-siRNA (TLR) were transfected into EBER-knockout EBV-infected AGS cells. After 48 h, cells were treated with EBER1 or poly(I:C) and IFN-β induction was analyzed by RT-PCR (bottom). Efficiency of TLR3 silencing was analyzed by RT-PCR (top). Three or more independent experiments were performed for each assay. (E) Effect of EBER1 on the downstream signals of TLR3, IRF3, and NF-κB. LCLs were treated with 2.5 µg/ml in vitro–synthesized EBER1 or poly(I:C) and cultured for 3 h before the phosphorylation of IRF3, and NF-κB was examined by immunoblotting using antibodies against phosphorylated IRF3, total IRF3, and phosphorylated p65. The data presented are representative of three independent experiments. (F) Effect of culture supernatants from EBER-positive cells on the expression of IFN-β. The study includes EBV-positive and –negative Mutu cells, EBV-positive and –negative Akata cells, and EBV-negative Akata cells that were stably infected with EBER-positive EBV or EBER-knockout EBV. The cells (2 × 105 cells/ml) were cultured for 4 d and then the culture supernatants were harvested. LCLs (4 × 105) were treated with 1 ml culture supernatants (top) or RNA extracted from 1 ml culture supernatants in 1 ml culture medium (bottom) for 14 h. RNA (0.1 µg) was subjected to 30 cycles of RT-PCR to detect IFN-β. Three or more independent experiments were performed for each assay.
Figure 3.
Figure 3.
EBER is detected as a complex with La in the culture supernatants and the complex is stimulatory for TLR3. (A) Detection of La, L22, and PKR in culture supernatants. Mutu+ cells (1 × 106) were transfected with Flag-tagged La, L22, or PKR plasmid and cultured for 48 h. Flag-tagged La, L22, and PKR were detected by immunoblotting of the cell lysates (top) and immunoprecipitation of culture supernatants (bottom) using an anti-Flag antibody. Three or more independent experiments were performed for each assay. (B) Detection of EBER1 in the immunoprecipitates. RNA was extracted from the immunoprecipitates and subjected to 30 cycles of RT-PCR to detect EBER1. Three independent experiments were performed. (C) Effect of immunoprecipitates from culture supernatants on IFN-β induction. EBER-positive (EBER+) or EBER-knockout (EBER−) EBV-infected AGS cells were transfected with control (control) or Flag-tagged La plasmid (Flag La) and cultured for 48 h, followed by immunoprecipitation of culture supernatants using anti-Flag antibody. Both EBER− and EBER+ immunoprecipitates were added into the media of EBER-knockout EBV-infected AGS cells transfected with negative control siRNA (Flag La+nc) or TLR3 siRNA (Flag La+TLR3si), and IFN-β induction was analyzed by RT-PCR. The data presented are representative of three independent experiments.
Figure 4.
Figure 4.
EBER1 exists in sera from patients with active EBV infections and induces the production of type I IFN and inflammatory cytokines. (A) Detection of EBER1 by RT-PCR in patient sera. RNA was extracted from 100 µl sera or plasma from patients with IM, CAEBV, and EBV-HLH, and from EBV-positive and –negative healthy donors. EBER1 was detected by 35 cycles of RT-PCR. Sample numbers in A–C correspond to each other; samples that are followed by a number in an open circle contain higher amounts of EBER1 than those with a number alone. Three or more independent experiments were performed. (B) Quantification of EBER1 in patient sera. RNA was extracted from the sera and subjected to real-time RT-PCR to detect EBER1. Error bars indicate the SD of duplicate wells. The data presented are representative of three independent experiments. (C) Induction of IFN-β production by RNA extracted from patient sera. LCLs (4 × 105) were treated with RNA that had been extracted from 100 µl sera in 1 ml culture medium, incubated for 14 h, and subjected to 30 cycles of RT-PCR to detect IFN-β (left) or ELISA of the culture supernatants for detection of IFN-β (right). The data of ELISA are shown as the means ± SD of duplicate determination and representative results of three independent experiments are shown. (D) Effect of an anti-TLR3 antibody on serum-induced IFN-β production. LCLs (4 × 105) were preincubated with the anti-TLR3 antibody for 30 min at 37°C, before being treated with RNA extracted from 100 µl of serum from a patient with IM or 1.0 µg in vitro–synthesized EBER1 as a positive control in 1 ml culture medium, and cultured for 14 h. Production of IFN-β was determined by ELISA of the culture supernatants. Error bars indicate the S.D. of duplicate wells. The data presented are representative of three independent experiments. (E) Effect of the RNA from patients sera on the induction of IFN-β and proinflammatory cytokines. Human PBMCs (1 × 106) were treated with RNA extracted from 100 µl patients sera or 0.5 µg in vitro–synthesized EBER1 in 1 ml culture medium and cultured for 14 h. The induction of IFN-β and proinflammatory cytokines was determined by 30 cycles of RT-PCR. Three independent experiments were performed.
Figure 5.
Figure 5.
EBER1 induces maturation of DC and subsequent antigen presentation. (A) Effect of EBER1 on phenotype of DCs. DCs were prepared from PBMCs and untreated or treated with either poly(I:C) (10 µg/ml) or EBER1 (10 µg/ml). Surface markers of matured DCs, CD83, and CD86 were measured by flow cytometry. As negative control, cells were stained with mouse IgG. The data are representative of three independent experiments. (B) Effect of TLR3 knockdown on EBER1-mediated DC maturation. DCs were transfected with TLR3 siRNA or control siRNA (nc) and were stimulated with EBER1. CD86 positivity (%) was analyzed by flow cytometry (left). Efficiency of TLR3 silencing was analyzed by RT-PCR (right). Data are shown as the means ± SD of duplicate determination and representative results of three independent experiments are shown. (C) EBER1-induced cytokine production by DCs. DCs were treated with EBER1, and IFN-β or IL-12p40 production were measured by ELISA. Error bars indicate the SD of duplicate wells, and the data presented are representative of three independent experiments. (D) Effect of sera from patients on TLR3-mediated IL-12 production by DCs. DCs were transfected with negative control siRNA or TLR3 siRNA (TLR3si) for 48 h, and then stimulated with sera from patients with CAEBV containing high amounts of EBER1 (CAEBV (1)) and IL-12 production was measured by ELISA. Sera form patients with CAEBV containing low amounts of EBER1 (CAEBV4) or EBV-negative (EBV(−)) healthy donor were also used for stimulation. Data are shown as the means ± SD of duplicate determination and representative results of three independent experiments are shown. (E) Allogenic MLR. DCs treated with either poly(I:C) or EBER1 were used as stimulator cells. Untreated immature DC were also used as stimulator cells. Allogenic PBMCs (1 × 105) were used as responder cells in triplicate cultures. Proliferation of alloreactive T cells was determined by cell proliferation assay. Data are shown as the means ± SD of triplicate determination and representative results of three independent experiments are shown. Statistical significance differences between groups were evaluated by Student's t test. ***, P < 0.001.
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