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Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5'-diphosphates.

Goubau D, Schlee M, Deddouche S, Pruijssers AJ, Zillinger T, Goldeck M, Schuberth C, Van der Veen AG, Fujimura T, Rehwinkel J, Iskarpatyoti JA, Barchet W, Ludwig J, Dermody TS, Hartmann G, Reis e Sousa C - Nature (2014)

Bottom Line: In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection.Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp.Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.

View Article: PubMed Central - PubMed

Affiliation: 1] Immunobiology Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK [2].

ABSTRACT
Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and β; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5'-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5'-diphosphates (5'pp). Genomes from mammalian reoviruses with 5'pp termini, 5'pp-RNA isolated from yeast L-A virus, and base-paired 5'pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5'pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.

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RNA from reovirus and L-A virus requires 5′-phosphates to induce a RIG-I-dependent response.(a-d) RNA samples were tested in an IFN-β promoter reporter assay in HEK293 cells: (a) RNA from reoT3D- or IAV-infected cells +/−CIP, (b) reoT3D vRNA +/−CIP, (c) reoT1L genome segments, and (d) reoT3D segments +/− CIP. For (b) and (d), RNA integrity was verified by gel electrophoresis. (e) IFN-α levels from transfected DCs. (f-h) IFN-α levels (f) or relative expression (RE) of ifit1 (g-h) from control (MDA5+/−), RIG-I−/−, MDA5−/− or RIG-I/MDA5−/− MEFs transfected with reo vRNA (f,h) or isolated reoT1L L segments (g) +/− CIP. Water and ppp-IVT-RNA99nts are controls. For (e-h), cells were treated with ribavirin to block virus replication. (i) Total L-A RNA (genome and transcript), L-A genomes and L-A transcripts were analysed as in (a). (j) Total L-A RNA was analysed as in (e). (k-l) Total L-A RNA +/− shrimp alkaline phosphatase (SAP) was analysed as in (a) or transfected into MDA5−/− DCs and analysed as in (g). Water, ppp-IVT-RNA99nts, poly(dA:dT) and cyclic-di-GMP were included as controls. All experiments were performed at least twice. For PCR and IFN-α data, the mean (±s.d.) of triplicate technical replicates is shown (* = not detected).
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Figure 1: RNA from reovirus and L-A virus requires 5′-phosphates to induce a RIG-I-dependent response.(a-d) RNA samples were tested in an IFN-β promoter reporter assay in HEK293 cells: (a) RNA from reoT3D- or IAV-infected cells +/−CIP, (b) reoT3D vRNA +/−CIP, (c) reoT1L genome segments, and (d) reoT3D segments +/− CIP. For (b) and (d), RNA integrity was verified by gel electrophoresis. (e) IFN-α levels from transfected DCs. (f-h) IFN-α levels (f) or relative expression (RE) of ifit1 (g-h) from control (MDA5+/−), RIG-I−/−, MDA5−/− or RIG-I/MDA5−/− MEFs transfected with reo vRNA (f,h) or isolated reoT1L L segments (g) +/− CIP. Water and ppp-IVT-RNA99nts are controls. For (e-h), cells were treated with ribavirin to block virus replication. (i) Total L-A RNA (genome and transcript), L-A genomes and L-A transcripts were analysed as in (a). (j) Total L-A RNA was analysed as in (e). (k-l) Total L-A RNA +/− shrimp alkaline phosphatase (SAP) was analysed as in (a) or transfected into MDA5−/− DCs and analysed as in (g). Water, ppp-IVT-RNA99nts, poly(dA:dT) and cyclic-di-GMP were included as controls. All experiments were performed at least twice. For PCR and IFN-α data, the mean (±s.d.) of triplicate technical replicates is shown (* = not detected).

Mentions: First, we assessed the 5′-phosphate-dependence of stimulation by reovirus RNA. RNA extracted from cells infected with reovirus strain type 3 Dearing (reoT3D) or isolated from reoT3D virus particles (viral RNA; vRNA) induced the expression of an IFN-β reporter gene following transfection into HEK293 cells (Fig. 1a, b). Calf intestinal phosphatase (CIP; Fig. 1a, b) treatment substantially reduced the stimulatory activity of reovirus vRNA, like it did of RNA from IAV-infected cells, a known 5′ppp-dependent RIG-I stimulus13,14. Similar results were obtained using reovirus strain type 1 Lang (reoT1L) and a distinct 5′-polyphosphatase (Extended data Fig. 1a and Supplementary Fig. 1i). The response to total reovirus vRNA could be recapitulated using purified large (L), medium (M), and small (S) genome segments (Fig. 1c, d and Extended data Fig. 1b) but not short (<20 residues, labeled <S; Fig. 1c) ssRNA oligonucleotides encapsidated within purified virions12.


Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5'-diphosphates.

Goubau D, Schlee M, Deddouche S, Pruijssers AJ, Zillinger T, Goldeck M, Schuberth C, Van der Veen AG, Fujimura T, Rehwinkel J, Iskarpatyoti JA, Barchet W, Ludwig J, Dermody TS, Hartmann G, Reis e Sousa C - Nature (2014)

RNA from reovirus and L-A virus requires 5′-phosphates to induce a RIG-I-dependent response.(a-d) RNA samples were tested in an IFN-β promoter reporter assay in HEK293 cells: (a) RNA from reoT3D- or IAV-infected cells +/−CIP, (b) reoT3D vRNA +/−CIP, (c) reoT1L genome segments, and (d) reoT3D segments +/− CIP. For (b) and (d), RNA integrity was verified by gel electrophoresis. (e) IFN-α levels from transfected DCs. (f-h) IFN-α levels (f) or relative expression (RE) of ifit1 (g-h) from control (MDA5+/−), RIG-I−/−, MDA5−/− or RIG-I/MDA5−/− MEFs transfected with reo vRNA (f,h) or isolated reoT1L L segments (g) +/− CIP. Water and ppp-IVT-RNA99nts are controls. For (e-h), cells were treated with ribavirin to block virus replication. (i) Total L-A RNA (genome and transcript), L-A genomes and L-A transcripts were analysed as in (a). (j) Total L-A RNA was analysed as in (e). (k-l) Total L-A RNA +/− shrimp alkaline phosphatase (SAP) was analysed as in (a) or transfected into MDA5−/− DCs and analysed as in (g). Water, ppp-IVT-RNA99nts, poly(dA:dT) and cyclic-di-GMP were included as controls. All experiments were performed at least twice. For PCR and IFN-α data, the mean (±s.d.) of triplicate technical replicates is shown (* = not detected).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4201573&req=5

Figure 1: RNA from reovirus and L-A virus requires 5′-phosphates to induce a RIG-I-dependent response.(a-d) RNA samples were tested in an IFN-β promoter reporter assay in HEK293 cells: (a) RNA from reoT3D- or IAV-infected cells +/−CIP, (b) reoT3D vRNA +/−CIP, (c) reoT1L genome segments, and (d) reoT3D segments +/− CIP. For (b) and (d), RNA integrity was verified by gel electrophoresis. (e) IFN-α levels from transfected DCs. (f-h) IFN-α levels (f) or relative expression (RE) of ifit1 (g-h) from control (MDA5+/−), RIG-I−/−, MDA5−/− or RIG-I/MDA5−/− MEFs transfected with reo vRNA (f,h) or isolated reoT1L L segments (g) +/− CIP. Water and ppp-IVT-RNA99nts are controls. For (e-h), cells were treated with ribavirin to block virus replication. (i) Total L-A RNA (genome and transcript), L-A genomes and L-A transcripts were analysed as in (a). (j) Total L-A RNA was analysed as in (e). (k-l) Total L-A RNA +/− shrimp alkaline phosphatase (SAP) was analysed as in (a) or transfected into MDA5−/− DCs and analysed as in (g). Water, ppp-IVT-RNA99nts, poly(dA:dT) and cyclic-di-GMP were included as controls. All experiments were performed at least twice. For PCR and IFN-α data, the mean (±s.d.) of triplicate technical replicates is shown (* = not detected).
Mentions: First, we assessed the 5′-phosphate-dependence of stimulation by reovirus RNA. RNA extracted from cells infected with reovirus strain type 3 Dearing (reoT3D) or isolated from reoT3D virus particles (viral RNA; vRNA) induced the expression of an IFN-β reporter gene following transfection into HEK293 cells (Fig. 1a, b). Calf intestinal phosphatase (CIP; Fig. 1a, b) treatment substantially reduced the stimulatory activity of reovirus vRNA, like it did of RNA from IAV-infected cells, a known 5′ppp-dependent RIG-I stimulus13,14. Similar results were obtained using reovirus strain type 1 Lang (reoT1L) and a distinct 5′-polyphosphatase (Extended data Fig. 1a and Supplementary Fig. 1i). The response to total reovirus vRNA could be recapitulated using purified large (L), medium (M), and small (S) genome segments (Fig. 1c, d and Extended data Fig. 1b) but not short (<20 residues, labeled <S; Fig. 1c) ssRNA oligonucleotides encapsidated within purified virions12.

Bottom Line: In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection.Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp.Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.

View Article: PubMed Central - PubMed

Affiliation: 1] Immunobiology Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK [2].

ABSTRACT
Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and β; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5'-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5'-diphosphates (5'pp). Genomes from mammalian reoviruses with 5'pp termini, 5'pp-RNA isolated from yeast L-A virus, and base-paired 5'pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5'pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.

Show MeSH
Related in: MedlinePlus