Small self-RNA generated by RNase L amplifies antiviral innate immunity

Abstract

Antiviral innate immunity is initiated in response to RNA molecules that are produced in virus-infected cells. These RNAs activate signalling cascades that activate the genes that encode alpha- and beta-interferon (IFN). Signalling occurs through the interaction of the RNAs with either of two pathogen recognition receptors, retinoic acid-inducible gene-I (RIG-I, also known as DDX58) and melanoma differentiation associated gene-5 (MDA5, also known as IFIH1), which contain amino-terminal caspase activation and recruitment domains (CARD) and carboxy-terminal DExD/H Box RNA helicase motifs. RIG-I and MDA5 interact with another CARD protein, interferon-beta promotor stimulator protein-1 (IPS-1, also known as MAVS, VISA and Cardif), in the mitochondrial membrane, which relays the signal through the transcription factors interferon regulatory factor 3 (IRF-3) and nuclear factor (NF)-kappaB to the IFN-beta gene. Although the signalling pathway is well understood, the origin of the RNA molecules that initiate these processes is not. Here we show that activation of the antiviral endoribonuclease, RNase L, by 2',5'-linked oligoadenylate (2-5A) produces small RNA cleavage products from self-RNA that initiate IFN production. Accordingly, mouse embryonic fibroblasts lacking RNase L were resistant to the induction of IFN-beta expression in response to 2-5A, dsRNA or viral infection. Single-stranded regions of RNA are cleaved 3' of UpUp and UpAp sequences by RNase L during viral infections, resulting in small, often duplex, RNAs. We show that small self-RNAs produced by the action of RNase L on cellular RNA induce IFN-beta expression and that the signalling involves RIG-I, MDA5 and IPS-1. Mice lacking RNase L produce significantly less IFN-beta during viral infections than infected wild-type mice. Furthermore, activation of RNase L with 2-5A in vivo induced the expression of IFN-beta in wild-type but not RNase L-deficient mice. Our results indicate that RNase L has an essential role in the innate antiviral immune response that relieves the requirement for direct sensing of non-self RNA.

Figure 1. Involvement of RNase L in induction of IFN-β expression by 2-5A, dsRNA or viral infection

a–c, IFN-β concentration from wild-type (WT) or RNase L-deficient MEFs that were mock transfected (Control) or transfected with (2′-5′)A₃ or (2′-5′) p₃A₃ for 16 h (a); mock transfected or transfected with poly(I):poly(C) for 18 h (b); or mock infected or infected with SeV for 18 h (c). d–f, IFN-β concentration from wild-type MEFs and RIG-I-deficient MEFs (d), MDA5-deficient MEFs (e) or IPS-1-deficient MEFs that were either mock transfected or transfected with unfractionated 2-5A for 16 h (f). Values are means from triplicate assays with standard deviations (s.d.).

Figure 2. 2-5A induces transcriptional activation of the IFN-β promoter

a, RIG-I, MDA5, IPS-1 and β-actin in DU145 cells treated for 48 h with siRNAs (shown above) in immunoblots. b, Activation of the IFN-β promoter in DU145 cells treated with siRNAs (5 nM) and 1.0 μM of (2′-5′)A₃ or (2′-5′)p₃A₃. c, Activation of the IFN-β promoter in Huh7 or Huh7.5 cells transfected with 1.0 μM of (2′-5′)A₃ or (2′-5′) p₃A₃ for 18 h. Relative luciferase (LUC) activity, firefly LUC/Renilla LUC. d, Stat1 phosphorylation in Huh7 or Huh7.5 cells treated with (2′-5′)p₃A₃ or (2′-5′)A₃ for 18 h. Stat1-P, phosphorylated Stat1; Stat1-T, total Stat1. Error bars, s.d.

Figure 3. Cleavage of cellular RNA by RNase L produces small RNAs that activate the IFN-β gene

a, RNA separated in RNA chip. b, c, IFN-β from MEFs transfected (for 18 h) with total (10 μg) RNA (b) or <200-nucleotide (nt) RNA (from 20 μg total RNA) (c). d, RIG-I (upper) and IRF-3 (lower) in RNase L-deficient MEFs and HT1080 cells, respectively, with <200-nt RNA (lanes 1–4) or wild-type MEFs (upper) or HT1080 cells (lower) infected with SeV (for 18 h) (lane 5). e–g, Levels of IFN-β from MEFs (wild-type, RIG-I-deficient, MDA5-deficient or IPS-1-deficient) incubated for 18 h with or without <200-nt RNA. Error bars, s.d.

Figure 4. RNase L contributes to induction of IFN-β by virus or 2-5A in vivo

Serum levels of IFN-β from RNase L-deficient or wild-type mice infected with EMCV (a) or SeV for the indicated times (b), or injected intraperitoneally with 2-5A or mock-treated for 5 h (c). Horizontal lines show mean levels of IFN-β. Two-tailed, paired Student’s t-tests were performed. d, Model for transcriptional activation of the IFN-β gene by small RNAs produced by cleavage of cellular (self) RNA with RNase L. Red, IFN-inducible proteins; 2′-PDE, 2′-phosphodiesterase; OAS, 2′-5′-oligoadenylate synthetase.