Alarm Bells: The presence of DNA in the cytosol of mammalian cells is a danger signal, indicating, for example, that a DNA-containing virus has infected the cell. This signal triggers an innate immune response, which involves the expression of type I interferons, and is critical for antiviral immunity and responses to DNA vaccines. Cyclic GMP-AMP synthase (cGAS) was recently identified as a sensor of cytosolic DNA. Li et al. (p. 1390 , published online 29 August) now use knockout mice to provide genetic evidence that, in multiple cell types, cGAS is the primary DNA sensor required for the type I interferon response in vivo.

Hepatitis C virus (HCV) is a global epidemic manifested mainly by chronic infection. One strategy that HCV employs to establish chronic infection is to use the viral Ser protease NS3/4A to cleave some unknown cellular targets involved in innate immunity. Here we show that the target of NS3/4A is the mitochondrial antiviral signaling protein, MAVS, that activates NF-κB and IFN regulatory factor 3 to induce type-I interferons. NS3/4A cleaves MAVS at Cys-508, resulting in the dislocation of the N-terminal fragment of MAVS from the mitochondria. Remarkably, a point mutation of MAVS at Cys-508 renders MAVS resistant to cleavage by NS3/4A, thus maintaining the ability of MAVS to induce interferons in HCV replicon cells. NS3/4A binds to and colocalizes with MAVS in the mitochondrial membrane, and it can cleave MAVS directly in vitro . These results provide an example of host–pathogen interaction in which the virus evades innate immunity by dislodging a pivotal antiviral protein from the mitochondria and suggest that blocking the cleavage of MAVS by NS3/4A may be applied to the prevention and treatment of HCV.

TREX1 is an exonuclease that digests DNA in the cytoplasm. Loss-of-function mutations of TREX1 are linked to Aicardi–Goutieres Syndrome (AGS) and systemic lupus erythematosus (SLE) in humans. Trex1^−/− mice exhibit autoimmune and inflammatory phenotypes that are associated with elevated expression of interferon (IFN)-induced genes (ISGs). Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates the IFN pathway. Upon binding to DNA, cGAS is activated to catalyze the synthesis of cGAMP, which functions as a second messenger that binds and activates the adaptor protein STING to induce IFNs and other cytokines. Here we show that genetic ablation of cGas in Trex1^−/− mice eliminated all detectable pathological and molecular phenotypes, including ISG induction, autoantibody production, aberrant T-cell activation, and lethality. Even deletion of just one allele of cGas largely rescued the phenotypes of Trex1^−/− mice. Similarly, deletion of cGas in mice lacking DNaseII, a lysosomal enzyme that digests DNA, rescued the lethal autoimmune phenotypes of the DNaseII^−/− mice. Through quantitative mass spectrometry, we found that cGAMP accumulated in mouse tissues deficient in Trex1 or DNaseII and that this accumulation was dependent on cGAS. These results demonstrate that cGAS activation causes the autoimmune diseases in Trex1^−/− and DNaseII^−/− mice and suggest that inhibition of cGAS may lead to prevention and treatment of some human autoimmune diseases caused by self-DNA. Significance The immune system detects microbial DNA in the cytosol of infected cells and mounts effective antimicrobial responses, including the production of type-I interferons. However, when self-DNA enters or accumulates in the cytosol, it can cause autoimmune diseases. Mutations of the exonuclease Trex1 in humans have been linked to autoimmune diseases including Aicardi–Goutieres Syndrome (AGS) and systemic lupus erythematosus (SLE). In mice, genetic deletion of Trex1 or the lysosomal nuclease DNaseII leads to lethal autoimmune diseases. Here we show that cyclic GMP-AMP synthase (cGAS) activation by self-DNA is responsible for the lethal autoimmune diseases in these models. These results provide the proof-of-concept that inhibition of cGAS may be an effective therapy for some autoimmune diseases such as AGS and SLE.

Modified vaccinia virus Ankara (MVA) is an attenuated poxvirus that has been engineered as a vaccine against infectious agents and cancers. Our goal is to understand how MVA modulates innate immunity in dendritic cells (DCs), which can provide insights to vaccine design. In this study, using murine bone marrow-derived dendritic cells, we assessed type I interferon (IFN) gene induction and protein secretion in response to MVA infection. We report that MVA infection elicits the production of type I IFN in murine conventional dendritic cells (cDCs), but not in plasmacytoid dendritic cells (pDCs). Transcription factors IRF3 (IFN regulatory factor 3) and IRF7, and the positive feedback loop mediated by IFNAR1 (IFN alpha/beta receptor 1), are required for the induction. MVA induction of type I IFN is fully dependent on STING (stimulator of IFN genes) and the newly discovered cytosolic DNA sensor cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase). MVA infection of cDCs triggers phosphorylation of TBK1 (Tank-binding kinase 1) and IRF3, which is abolished in the absence of cGAS and STING. Furthermore, intravenous delivery of MVA induces type I IFN in wild-type mice, but not in mice lacking STING or IRF3. Treatment of cDCs with inhibitors of endosomal and lysosomal acidification or the lysosomal enzyme Cathepsin B attenuated MVA-induced type I IFN production, indicating that lysosomal enzymatic processing of virions is important for MVA sensing. Taken together, our results demonstrate a critical role of the cGAS/STING-mediated cytosolic DNA-sensing pathway for type I IFN induction in cDCs by MVA. We present evidence that vaccinia virulence factors E3 and N1 inhibit the activation of IRF3 and the induction of IFNB gene in MVA-infected cDCs. Modified vaccinia virus Ankara (MVA) is an attenuated vaccinia strain with large deletions of the parental genome that render it non-replicative in mammalian cells. MVA is a safe and effective vaccine against both smallpox and monkeypox. MVA has been investigated as a vaccine vector for infectious diseases and cancers. Dendritic cells (DCs) play important roles in innate and adaptive immunity. A better understanding of how MVA is detected by innate immune sensors in DCs would guide the development of more effective MVA-based vaccines. We report our findings that MVA infection induces the production of type I interferon (IFN) in conventional dendritic cells via a cytosolic DNA-sensing pathway mediated by the newly discovered DNA sensor cGAS, its adaptor STING, and transcription factors IRF3 and IRF7. By contrast, wild-type vaccinia virus fails to activate this pathway. Furthermore, we show that vaccinia virulence factors E3 and N1 play inhibitory roles in the cytosolic DNA-sensing pathway.