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

Abstract

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 Burkitt's 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-I's substrates, NF-kappaB 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.

Figure 1

RIG-I induces type I IFN in EBV-infected cells. EBV-positive Mutu cells (Mutu(+)) and EBV-negative Mutu cells (Mutu(−)) (5 × 10⁶ each) were transfected with 30 μg of GFP-tagged RIG-I plasmid or control GFP plasmid, and expression of IFNs and ISG56 was examined at the designated time by RT–PCR. (A) Immunoblot analysis for detection of RIG-I. The blots were probed with anti-RIG-I antibody (left panel) and anti-GFP antibody (right panel). Cell lysates were prepared after 24 h of transfection and 20 μg of protein sample was loaded per slot. (B) RT–PCR analysis of expression of IFN-α, IFN-β, and ISG56 in Mutu (+) cells. (C) RT–PCR analysis of expression of IFN-α, IFN-β, and ISG56 in Mutu (−) cells. (D) RT–PCR analysis of EBER1 expression in Mutu(+) and Mutu(−) cells.

Figure 2

RIG-I induces type I IFN in EBER-expressing cells. BL-derived, EBER-positive and EBER-negative Akata cells (5 × 10⁶ each) were transfected with 30 μg of GFP-tagged RIG-I plasmid or GFP plasmid, and expression of IFN-β and ISG56 was examined at the designated time by RT–PCR. (A) Expression of IFN-β and ISG56 in Akata cells infected with EBER-knockout EBV (EBER(−) EBV) or EBER-reintroduced EBV (EBER(+) EBV). After 12 h of transfection of the RIG-I plasmid or control plasmid, cells were subjected to RT–PCR analysis. (B) Expression of IFN-β and ISG56 in Akata cells stably transfected with EBER plasmid (Akt/EBER) or in those with control plasmid carrying the neomycin resistance gene (Akt/Neo^r). After 6 and 12 h of transfection of the RIG-I plasmid or GFP plasmid, cells were subjected to RT–PCR analysis. (C) EBER-positive EBV-infected and EBER-negative EBV-infected Akata cells were transfected with 100 nM of RIG-I siRNA or control siRNA. After 24 h, expression of RIG-I and IFN-β was checked by RT–PCR. (D) EBER-expressing Akata cells and control cells were also transfected with 100 nM of RIG-I siRNA/control siRNA and the expression of RIG-I and IFN-β was examined at 24 h post-transfection.

Figure 3

EBERs induce type I IFN in RIG-I-expressing cell clones. EBV-negative Daudi cells were transfected with the plasmid carrying GFP-tagged RIG-I or control plasmid carrying the GFP gene only, and cell clones stably expressing RIG-I/GFP or GFP were isolated. They (5 × 10⁶ each) were transfected with 30 μg of EBER plasmid, and expression of type I IFN was examined at the designated time by RT–PCR. (A) Immunoblot analysis for detection of RIG-I. The blots were probed with the anti-GFP antibody. Protein samples amounting to 20 μg was loaded per slot. (B) RT–PCR analysis for detection of IFN-β.

Figure 4

Both EBER1 and EBER2 activate RIG-I to induce type I IFN. (A) Analysis of in vitro-synthesized EBER1 and EBER2 in 5% denaturing PAGE before (left panel) and after (middle panel) RNase A treatment. RT–PCR analysis of EBER1 cDNA with EBER1 and EBER2 primers and also EBER2 cDNA with EBER2 and EBER1 primers (right panel). (B) RT–PCR analysis of expression of IFN-β, ISG56, and ISG15 after transfection of EBER1 and EBER2. RIG-I- or GFP- (5 × 10⁶ each) expressing stable clones were transfected with 30 μg of EBER1 or EBER2, separately or in combination (1:1). Expression of IFN-β and ISGs was examined after 6 and 24 h of transfection.

Figure 5

EBERs activate NF-κB and IRF-3. (A) Phosphorylation of NF-κB in RIG-I- or GFP-expressing stable clone was analyzed after they (5 × 10⁶ cells each) were transfected with 30 μg of in vitro-synthesized EBERs (1:1) or polyI:C (positive control). After 6 h of transfection, whole-cell lysates (20 μg of protein for each sample) were subjected to immunoblot analysis for detection of phosphorylated NF-κB. (B) NF-κB reporter assay. NF-κB plasmid was transfected into both RIG-I-expressing Daudi cell clone and control clone and after 36 h of transfection, EBERs or polyI:C were transfected. After 12 h of EBER or polyI:C transfection, luciferase activity was measured in cell lysates (Figure 5B). Results are shown as the means±standard errors from three independent experiments. (C) Effect of IκB-α, a specific inhibitor of NF-κB, on EBER-induced phosphorylation of NF-κB. RIG-I-expressing stable clones (5 × 10⁶ cells) were first transfected with 10 μg (+) or 20 μg (++) of IκB-α plasmid. After 36 h, they were further transfected with 30 μg of in vitro-synthesized EBERs (1:1), and expression of IFN-β was examined by RT–PCR after 8 h of transfection. (D) Inhibition of NF-κB activation by IκB-α plasmid. RIG-I-expressing stable clones (5 × 10⁶ cells) were transfected with 10 μg of pNF-κB/luc (firefly luciferase) and 100 ng of pCMV/luc (Renilla luciferase as an internal control), along with 10 μg (+) or 20 μg (++) of IκB-α plasmid. After 36 h, luciferase activity was quantified in cell lysates using a Dual-Luciferase reporter assay system (Promega). Results are shown as the means±standard errors from three independent experiments. (E) Phosphorylation of IRF-3 in EBV-negative Daudi cells stably expressing RIG-I. RIG-I- or GFP- (5 × 10⁶ cells each) expressing stable clones were transfected with 30 μg of in vitro-synthesized EBERs (15 μg each) or polyI:C (positive control). After 2 and 6 h of transfection, whole-cell lysates (30 μg of protein for each sample) were separated with native PAGE gel (8%) to analyze the phosphorylation of IRF-3 by EBERs or polyI:C. The blot was probed with anti-phosho-IRF-3 antibody. This antibody reacted with phosphorylated IRF-3, but not with unphosphorylated IRF-3. (F) RIG-I-expressing stable clone was treated with 100 nM of IRF-3 siRNA or control siRNA. After 24 h, cells were transfected with 30 μg of in vitro-synthesized EBERs, and 6 h later, expression of IRF-3, IFN-β, and ISG56 was examined by RT–PCR.

Figure 6

EBER is recognized by full-length RIG-I. (A) Schematic diagram of mutant plasmids containing the CARD domain (1–284 aa) or the helicase domain (218–925 aa). (B) Immunoblot analysis for detection of mutant RIG-I expressions in EBV-negative Daudi cell clones stably transfected with the deletion plasmids containing GFP-tagged CARD domain (N-RIG) or GFP-tagged helicase domain (C-RIG). Expression of N-RIG and C-RIG was detected by anti-RIG-I antibody (left panel) and also after reprobing with anti-GFP antibody (right panel). (C) Expression of type I IFN and ISGs in EBV-negative Daudi cells stably transfected with the deletion plasmids of RIG-I. The cells (5 × 10⁶ cells each) were transfected with 30 μg of EBERs or polyI:C, and after 6 h, subjected to RT–PCR for detection of IFN-β, ISG15, and ISG56.

Figure 7

Binding assay for detection of association of EBERs with RIG-I. The full-length RIG-I plasmid (RIG-I/GFP) or control plasmid (containing the GFP gene only) was transfected into EBV-negative Mutu cells (Mutu(−) cells), EBER-positive EBV-infected Mutu cells ((Mutu(+) cells), and EBER-transfected Mutu cells (Mutu/EBER cells). After 48 h, cells were treated with UV irradiation, digested with RNases, and subjected to immunoprecipitation with the anti-GFP antibody. RNA was isolated from immunoprecipitants, and EBER1 and EBER2 were measured by RT–PCR.

Figure 8

RIG-I recognizes EBERs in the early phase of EBV infection. EBV-negative Daudi cell clones stably expressing RIG-I/GFP or GFP were infected with EBER(+) EBV or EBER(−) EBV, and expression of EBER1, EBNA2, and IFN-β was examined at the designated time of EBV infection by RT–PCR.