Detection of cytoplasmic DNA represents one of the most fundamental mechanisms of the innate immune system to sense the presence of microbial pathogens1. Moreover, erroneous detection of endogenous DNA by the same sensing mechanisms has an important pathophysiological role in certain sterile inflammatory conditions2, 3. The endoplasmic-reticulum-resident protein STING is critically required for the initiation of type I interferon signalling upon detection of cytosolic DNA of both exogenous and endogenous origin4, 5, 6, 7, 8. Next to its pivotal role in DNA sensing, STING also serves as a direct receptor for the detection of cyclic dinucleotides, which function as second messenger molecules in bacteria9, 10, 11, 12, 13. DNA recognition, however, is triggered in an indirect fashion that depends on a recently characterized cytoplasmic nucleotidyl transferase, termed cGAMP synthase (cGAS), which upon interaction with DNA synthesizes a dinucleotide molecule that in turn binds to and activates STING14, 15. We here show in vivo and in vitro that the cGAS-catalysed reaction product is distinct from previously characterized cyclic dinucleotides. Using a combinatorial approach based on mass spectrometry, enzymatic digestion, NMR analysis and chemical synthesis we demonstrate that cGAS produces a cyclic GMP-AMP dinucleotide, which comprises a 2′-5′ and a 3′-5′ phosphodiester linkage >Gp(2′-5′)Ap(3′-5′)>. We found that the presence of this 2′-5′ linkage was required to exert potent activation of human STING. Moreover, we show that cGAS first catalyses the synthesis of a linear 2′-5′-linked dinucleotide, which is then subject to cGAS-dependent cyclization in a second step through a 3′-5′ phosphodiester linkage. This 13-membered ring structure defines a novel class of second messenger molecules, extending the family of 2′-5′-linked antiviral biomolecules.

SUMMARY 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 infection1,2. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α/β; hereafter IFN), 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 1-3). 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 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.

RIG-I is a cytosolic helicase that senses 5’-ppp-RNA contained in negative strand RNA viruses and triggers innate antiviral immune responses. Calorimetric binding studies establish that the RIG-I C-terminal regulatory domain (CTD) binds to blunt-end double-stranded 5’-ppp-RNA a factor of 17 more tightly than to its single-stranded counterpart. Here we report on the crystal structure of RIG-I CTD domain bound to both blunt-ends of a self-complementary 5’-ppp-dsRNA 12-mer, with interactions involving 5’-pp clearly visible in the complex. The structure, supported by mutation studies, defines how a lysine-rich basic cleft within the RIG-I CTD domain sequesters the observable 5’-pp of the bound RNA, with a stacked Phe capping the terminal base pair. Key intermolecular interactions observed in the crystalline state are retained in the complex of 5’-ppp-dsRNA 24-mer and full-length RIG-I under in vivo conditions, as evaluated from the impact of binding pocket RIG-I mutations and 2’-OCH3 RNA modifications on the interferon response.

The cytosolic helicase retinoic acid-inducible gene-I (RIG-I) initiates immune responses to most RNA viruses by detecting viral 5'-triphosphorylated RNA (pppRNA). Although endogenous mRNA is also 5'-triphosphorylated, backbone modifications and the 5'-ppp-linked methylguanosine ((m7)G) cap prevent immunorecognition. Here we show that the methylation status of endogenous capped mRNA at the 5'-terminal nucleotide (N1) was crucial to prevent RIG-I activation. Moreover, we identified a single conserved amino acid (H830) in the RIG-I RNA binding pocket as the mediator of steric exclusion of N1-2'O-methylated RNA. H830A alteration (RIG-I(H830A)) restored binding of N1-2'O-methylated pppRNA. Consequently, endogenous mRNA activated the RIG-I(H830A) mutant but not wild-type RIG-I. Similarly, knockdown of the endogenous N1-2'O-methyltransferase led to considerable RIG-I stimulation in the absence of exogenous stimuli. Studies involving yellow-fever-virus-encoded 2'O-methyltransferase and RIG-I(H830A) revealed that viruses exploit this mechanism to escape RIG-I. Our data reveal a new role for cap N1-2'O-methylation in RIG-I tolerance of self-RNA.

Intracellular recognition of non-self and also self-nucleic acids can result in the initiation of potent pro-inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2 0 -5 0 ), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP-1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS-STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA.

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon–inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

Two virulence factors produced by Pseudomonas aeruginosa are pyocyanin and N-(3-oxododecanoyl)-L-homoserine lactone (3OC12). Pyocyanin damages host cells by generating ROS (reactive oxygen species). 3OC12 is a quorum-sensing signalling molecule which regulates bacterial gene expression and modulates host immune responses. PON2 (paraoxonase-2) is an esterase that inactivates 3OC12 and potentially attenuates Ps. aeruginosa virulence. Because increased intracellular Ca2+ initiates the degradation of PON2 mRNA and protein and 3OC12 causes increases in cytosolic Ca2+, we hypothesized that 3OC12 would also down-regulate PON2. 3OC12 and the Ca2+ ionophore A23187 caused a rapid cytosolic Ca2+ influx and down-regulated PON2 mRNA, protein and hydrolytic activity in A549 and EA.hy 926 cells. The decrease in PON2 hydrolytic activity was much more extensive and rapid than decreases in protein, suggesting a rapid post-translational mechanism which blocks PON2's hydrolytic activity. The Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] diminished the ability of 3OC12 to decrease PON2, demonstrating that the effects are mediated by Ca2+. PON2 also has antioxidative properties and we show that it protects cells from pyocyanin-induced oxidative stress. Knockdown of PON2 by transfecting cells with siRNA (small interfering RNA) rendered them more sensitive to, whereas overexpression of PON2 protected cells from, pyocyanin-induced ROS formation. Additionally, 3OC12 potentiated pyocyanin-induced ROS formation, presumably by inactivating PON2. These findings support a key role for PON2 in the defence against Ps. aeruginosa virulence, but also reveal a mechanism by which the bacterium may subvert the protection afforded by PON2.

The cytosolic antiviral innate immune sensor RIG-I distinguishes 5′ tri- or diphosphate containing viral double-stranded (ds) RNA from self-RNA by an incompletely understood mechanism that involves ATP hydrolysis by RIG-I's RNA translocase domain. Recently discovered mutations in ATPase motifs can lead to the multi-system disorder Singleton-Merten Syndrome (SMS) and increased interferon levels, suggesting misregulated signaling by RIG-I. Here we report that SMS mutations phenocopy a mutation that allows ATP binding but prevents hydrolysis. ATPase deficient RIG-I constitutively signals through endogenous RNA and co-purifies with self-RNA even from virus infected cells. Biochemical studies and cryo-electron microscopy identify a 60S ribosomal expansion segment as a dominant self-RNA that is stably bound by ATPase deficient RIG-I. ATP hydrolysis displaces wild-type RIG-I from this self-RNA but not from 5' triphosphate dsRNA. Our results indicate that ATP-hydrolysis prevents recognition of self-RNA and suggest that SMS mutations lead to unintentional signaling through prolonged RNA binding.DOI: http://dx.doi.org/10.7554/eLife.10859.001