TLR3 ACTIVATION BY EBV-ENCODED SMALL RNA

Iwakiri D, Zhou L, Samanta M, Matsumoto M, Ebihara T, Seya T, Imai S, Fujieda M, Kawa K, Takada K.

Epstein-Barr virus (EBV)-encoded small RNA is released from EBV-infected cells and activates

signaling from Toll-like receptor 3. J Exp Med. 2009 Sep 28;206(10):2091-9. Epub 2009 Aug 31.

PubMed PMID: 19720839; PubMed Central PMCID: PMC2757889.

 

Epstein-Barr- virus encoded small RNA (EBER) is nonpolyadenylated, noncoding RNA that forms stem-loop structure by base pairing and giving rise to double-stranded RNA (dsRNA)-like molecules. EBER1 and EBER2 are 167 and 172 nucleotide long. EBER is the most abundant viral RNA in the latently infected cells. It binds to several cellular proteins including RNA activated protein kinase, ribosomal protein 22, lupus erythematosis-associated antigen and retinoic acid-inducible gene I.

In this interesting article, the investigators found that EBER exists in the sera of patients with active EBV infection and induces type I IFN and inflammatory cytokines through TLR-3 mediated signaling. The authors postulate that this may account for the pathogenesis of active EBV infections that are characterized by cytokinemia.

RESULTS:

• EBER is present in the culture supernatants of EBV-infected cells: The authors performed RT-PCR analysis of EBER1 in culture supernatants of Burkitt's lymphoma derived EBV-infected cell lines (Mutu+, Akata+) and EBV transformed lymphoblastoid cell lines (LCLs). They observed that EBER was detected at day 1 of culture and its expression increased and peaked on day 4. The release of EBER1 was higher than EBER2.
  

  
   

• EBER induces signaling from TLR3:
  
  • To investigate the role of EBER released from EBV infected cells, the authors first examined whether in vitro synthesized EBER1 could induce signaling from TLR3. They subjected total RNA from three LCL clones and EBV-infected and uninfected NU-GC-3 cells to RT PCR to detect TLR3. The results indicated that LCLs and gastric carcinoma NU-GC-3 cells expressed TLR3.
    
  • The authors then added in vitro synthesized EBER1 into culture medium and found the induction of IFN-beta in LCLs and EBV positive and negative NU-GC-3 cells. The authors performed similar experiments using poly (I:C) instead of EBER and observed similar results. On performing ELISA, the authors observed that IFN-beta production was dependent on the amount of EBER1 that was added to the culture.
    
  • Furthermore, when the authors pretreated LCLs with an anti-TLR3 antibody and then with EBER1, the expression of IFN-beta was markedly reduced as observed by both RT PCR and ELISA.
    
  • The authors then looked for the effect of TLR-3 knockdown on EBER-1 induced IFN-beta production. For this, negative control siRNA or TLR-3-siRNA were transfected into EBER knockout-EBV infected AGS cells and after 48 hrs, these cells were treated with EBER1 or poly (I:C) and IFN-beta production was analyzed by RT PCR. TLR3 knockout caused reduced induction of IFN. These results indicated that EBER1 induces IFN through TLR3.
    
  • IFN-regulatory factor 3 (IRF-3) and NF-kappa B function downstream of TLR3 signaling pathway. To analyze the effect of in vitro EBER1 on these downstream signals, the authors treated LCLs with in vitro synthesized EBER1 or poly(I:C) and cultured for 3 hrs after which they examined the phosphorylation of IRF3 and NF-kappa B by immunoblotting using antibodies against phosphorylated IRF3, total IRF3 and phosphorylated p65. They observed that both IRF-3 and NF-kappa B were phosphorylated upon treatment of the cells with EBER1 and poly(I:C).
    
  • Now, the authors examined whether EBER1 that was released in the culture supernatant could induce the expression of IFN. EBV positive and EBV negative Mutu cells, EBV-positive and EBV-negative Akata cells, and EBV-negative Akata cells that were stably infected with EBER-positive EBV or EBER-knockout EBV were cultured for 4 days and then the culture supernatants were harvested. LCLs were treated with 1ml of these culture supernatants or with RNA extracted from 1ml culture supernatant. The RNA extracted from the LCLs was subjected to RT-PCR to detect IFN-beta. The results indicated that IFN was induced in EBV-positive cells but not in EBV-negative cells or EBER-knockout EBV-infected cells.
    

    
     

• EBER1 is detected as a complex with La in the culture supernatants, and the complex can induce TLR3 signaling: The previous results demonstrated that EER1 was stably present in the culture supernatants which meant that it must be bound by some proteins and thus is protected from degradation by nucleases. The investigators therefore examined whether EBER1 existed as a complex with EBER-binding cellular proteins in culture supernatants. They transfected Mutu+ cells with Flag-tagged La, L22 and PKR expression plasmids, cultured them for 48 hrs and detected La, L22 and PKR by immunoblotting of the cell lysates and immunoprecipitation of culture supernatants using anti-Flag antibody. They observed that although Flag-tagged L22, La and PKR were expressed equally in transfected cells, only La could be strongly immunoprecipitated from the culture supernatant. The authors then detected EBER1 in the immunoprecipitates using RT PCR. For this, RNA was extracted from the immunoprecipitates and subjected to RT-PCR to detect EBER1. The results showed that EBER1 was preferentially co-precipitated with La. The presence of La in the culture supernatants indicated that La was actively secreted from the living cells rather than passively released from dead cells. The results further suggested that EBER1 is released from EBV-infected cells as a complex with La. To further investigate whether EBER can activate TLR3 in complexes with La, they transfected EBER positive- and EBER knockout- EBV infected AGS cells with Flag-tagged La and cultured for 48 hrs. The culture supernatants were immunoprecipitated with anti-Flag antibody. Both these EBER+ and EBER- immunoprecipitates were added into the media of EBER-knockout EBV-infected AGS cells previously transfected with TLR3 siRNA (TLR3 knockdown) or with control siRNA. IFN-beta induction was analyzed by RT PCR. IFN-beta induction was clearly induced by treatment with EBER+ immunoprecipitates, while no induction was seen upon treatment with immunoprecipitates from EBER negative cells. The induction was reduced by TLR3 knockdown. These results indicate that EBER1 can induce TLR3 signaling in complexes with La.
  

  
   

• EBER1 exists in sera from patients with active EBV infections and induces the production of type I IFN and inflammatory cytokines: The investigators next extracted RNA from sera or plasma of patients with infectious mononucleosis (IM), chronic active EBV infection (CAEBV), EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and EBV positive and negative healthy donors and subjected to RT PCR for detection of EBER1. Results showed that EBER1 was detected in patient sera and in sera of healthy individuals. The level of EBER1 was much higher in patient sera than in sera of healthy individuals. No EBER1 was detected in sera of EBV negative healthy individuals. Furthermore, LCLs were treated with this RNA, incubated for 14hrs and subjected to RT PCR to detect IFN-beta and ELISA of the culture supernatant. The results showed that RNAs from those patient sera that contained a high amount of EBER1, induced the release of IFN-beta when added to the culture medium of LCLs. To confirm that IFN-beta production by patient sera was mediated through TLR3, the authors treated LCLs with anti-TLR3 anitbody before subjecting them to RNA from sera of patient. A marked reduction in the RNA-induced release of IFN-beta was observed. These results clearly indicated that RNA from serum induced the expression of IFN-beta through TLR3. The authors also tend to determine whether RNA from patients sera can cause induction of proinflammatory cytokines. Thus, they treated human PBMCs with RNA extracted from patients sera or in vitro synthesized EBER1 and cultured for 14hrs. Induction of IFN-gamma, and TNF-alpha was observed by RNA extracted from patient sera as well as by in vitro EBER1.
  

  
   

• EBER1 induces mature surface phenotype and antigen presenting capacity of DCs: To study the effect of EBER1 on DC phenotype, the investigators treated immature DCs with either poly(I:C) or EBER1 for 24 hrs and analyzed the surface markers of matured DCs by flow cytometry. They found that both the treatments caused increased levels of CD83 and CD86, indicating that EBER1 induces maturation of DCs. The treatment with si-TLR3 caused reduction in the up-regulation of CD86, indicating that EBER1 mediated DC maturation is TLR dependent. Next, the authors treated DCs with EBER1 and analyzed the production of IFN-beta and IL-12p40. The results indicated that IFN-beta and IL-12 production by DC was induced by EBER1 and thus EBER causes activation of DCs. To assess whether this activation of DCs by EBER is dependent on TLR3 or not, DCs were transfected with negative control siRNA or siRNA-TLR3 and then stimulated with sera from patients CAEBV containing high amounts of EBER1, low amounts of EBER1 and EBV-negative patients and IL-12 production was determined. The results indicated that the IL-12 production by DCs occurs through EBER1 mediated TLR3 signaling. To further determine whether DCs matured by EBER1 have the capacity for antigen presentation, the DCs treated with EBER1 or poly(I:C) were used for alloMLR assay. Here the authors compared the stimulatory properties of EBER1 or poly (I:C) treated DCs with those of untreated DCs. In this assay, after 24hr stimulation with EBER1 or poly(I:C), DCs were treated with mitomycin. Allo PBMCs were isolated from blood of healthy donors and cultured with mitomycin treated DCs. The PBMC proliferation was then measured using CellTiter 96 nonradioactive cell proliferation assay kit. The results indicated that EBER1-treated DCs were potent inducers of primary allo T cell responses.