Innate immunity is the first line of host defense against microbial pathogens. Host cells utilize their pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs). The Toll-like receptor family (TLRs) is one class of PRRs that recognize PAMPs including lipoproteins, lipopolysaccharides (LPS), double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), and unmethylated CpG DNA (reviewed by Akira et al., 2006
). Ligand-engaged TLRs recruit the adaptor proteins MyD88 or TRIF to activate downstream kinases including IκB kinase complex (IKK) and IKK-related kinases (TBK1 and IKKε), which activate the transcription factors nuclear factor-kappa B (NF-κB) and interferon regulatory factors (IRFs), respectively. NF-κB and IRFs function together in the nucleus to induce type-I interferons (IFNs; e.g., IFN-α and IFN-β) and other cytokines.
In another PRR pathway, cytosolic RNAs are recognized by RIG-I-like receptors (RLRs), which include RIG-I, MDA5 and LGP2 (Yoneyama et al., 2004
). RLRs contain RNA helicase domains that recognize viral double-stranded RNA. In addition, RIG-I and LGP2 contain a C-terminal regulatory domain that recognizes single-stranded RNA containing 5′-triphosphate, which distinguishes foreign (e.g, viral) RNAs from self-RNAs that normally contain 5′-modification (e.g, capped mRNA). RIG-I and MDA5 also contain N-terminal tandem caspase activation and recruitment domains (CARD), which interact with the CARD domain of mitochondrial antiviral signaling protein (MAVS, also known as IPS-1, VISA and CARDIF) (Kawai et al., 2005
; Meylan et al., 2005
; Seth et al., 2005
; Xu et al., 2005
). MAVS localizes on the mitochondrial outer membrane through its C-terminal transmembrane domain, and this localization is important for MAVS to activate the cytosolic kinases IKK and TBK1 to induce IFNs (Seth et al., 2005
Like RNA, accumulation of foreign or self DNA in the cytosol also triggers potent innate immune responses. DNA can be introduced into the cytosol of mammalian cells following infection with DNA viruses or bacteria, and the detection of cytosolic DNA is important for mounting an immune response against these pathogens. Under certain conditions, self DNA is inappropriately delivered to the cytosol, resulting in autoimmune responses. For example, DNase II-deficient macrophages lack the ability to digest self-DNA from engulfed apoptotic cells, leading to IFN-β production (Okabe et al., 2005
; Yoshida et al., 2005
). However, the mechanism by which cytosolic DNA induces IFNs is not well understood. In particular, the sensor that detects cytosolic DNA and triggers IFN production has remained largely unknown. Although DNA-dependent activator of IFN-regulatory factors (DAI) has been proposed to be a potential cytosolic DNA sensor (Takaoka et al., 2007
), DAI-deficient mice still produce interferons in response to B-form DNA and have similar innate and adaptive immune responses to those of wild-type mice (Ishii et al., 2008
). Recent studies identify AIM2 as a cytosolic DNA sensor that activates the inflammasome and caspase-1 (reviewed by Schroder et al., 2009
). However, AIM2 is not involved in type-I interferon induction by cytosolic DNA.
Genetic studies have shown that cytosolic DNA can induce IFN production in mouse cells lacking RIG-I or MAVS, suggesting that the DNA signaling pathway is distinct from the RIG-I pathway (Ishii et al., 2006
; Sun et al., 2006
). Nevertheless, there is evidence that in certain human cell lines the induction of IFN-β by transfected double-stranded DNA depends on RIG-I and MAVS (Cheng et al., 2007
; Ishii et al., 2006
). However, RIG-I binds to RNA but not DNA, raising the question of how DNA might activate the RIG-I pathway.
In this report, we show that the double-stranded DNA poly(dA-dT)·poly(dA-dT), herein referred to as poly(dA-dT), is converted to an RNA species in the cytosol to trigger the RIG-I pathway in human and mouse cells. This RNA species contains 5′-triphosphate and forms a double-stranded RNA. The conversion of DNA to RNA can be recapitulated in vitro using cytosolic extracts. Biochemical purification led to the identification of DNA-dependent RNA polymerase III (Pol-III) as the enzyme responsible for transcribing the DNA template into an RNA ligand that activates RIG-I. RNAi-mediated knock down of Pol-III expression or inhibition of its enzymatic activity impedes interferon induction by transfection of DNA or infection with several DNA viruses, including adenovirus, herpes simplex virus 1 (HSV-1), and Epstein-Barr virus (EBV). Moreover, Pol-III inhibition blocks interferon induction by the intracellular bacterium Legionella pneumophila. These results strongly suggest that Pol-III is a cytosolic DNA sensor that triggers type-I interferon production through the RIG-I pathway.