The detection of intracellular microbial DNA is critical to an appropriate innate immune response, however current knowledge on how such DNA is sensed is limited. Here we identify IFI16, a PYHIN protein, as an intracellular DNA sensor mediating interferon-β (IFNβ)-induction. IFI16 directly associated with IFNβ-inducing viral DNA motifs. STING, a critical mediator of IFNβ responses to DNA, was recruited to IFI16 after DNA stimulation. Reduction of expression of IFI16, or its murine ortholog p204, by RNA interference inhibited DNA- and herpes simplex virus (HSV)-1-induced gene induction and IRF3 and NFκB activation. IFI16/p204 is the first PYHIN protein shown to be involved in IFNβ induction, and thus together with AIM2, a PYHIN protein that senses DNA for caspase 1 activation, is part of a new family of innate DNA sensors which we term AIM2-like receptors (ALRs).
Viruses are detected by different classes of pattern recognition receptors (PRRs), such as Toll-like receptors and RIG-like helicases. Engagement of PRRs leads to activation of interferon (IFN)-regulatory factor 3 (IRF3) and IRF7 through IKKe and TBK1 and consequently IFN-b induction. Vaccinia virus (VACV) encodes proteins that manipulate host signalling, sometimes by targeting uncharacterised proteins. Here, we describe a novel VACV protein, K7, which can inhibit PRR-induced IFN-b induction by preventing TBK1/IKKe-mediated IRF activation. We identified DEAD box protein 3 (DDX3) as a host target of K7. Expression of DDX3 enhanced Ifnb promoter induction by TBK1/IKKe, whereas knockdown of DDX3 inhibited this, and virus-or dsRNA-induced IRF3 activation. Further, dominant-negative DDX3 inhibited virus-, dsRNA-and cytosolic DNA-stimulated Ccl5 promoter induction, which is also TBK1/IKKe dependent. Both K7 binding and enhancement of Ifnb induction mapped to the N-terminus of DDX3. Furthermore, virus infection induced an association between DDX3 and IKKe. Therefore, this study shows for the first time the involvement of a DEAD box helicase in TBK1/IKKe-mediated IRF activation and Ifnb promoter induction.
Recognition of viruses by pattern recognition receptors (PRRs) causes interferon-β (IFN-β) induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV) protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs) to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1) and IκB kinase-ε (IKKε), which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.
The detection of nucleic acids by the innate immune system is an essential host response during viral infection. In recent years, a number of immune sensors capable of recognizing cytosolic DNA have been identified and include the PYHIN family members AIM2, IFI16, and p204 as well as the enzyme, cGAS. Activation of these receptors leads to the induction of antiviral genes including Type‐1 interferons and chemokines such as CCL5. We have carried out extensive expression profiling of these DNA sensors and other members of the PYHIN family in highly purified primary astrocytes and microglia and have demonstrated that both cell types express the majority of these proteins at the mRNA level. In microglia, several family members are highly upregulated in response to IFN‐β treatment while both cell types induce robust proinflammatory and antiviral cytokine production (e.g., IL‐6, CCL5, IFN‐β) in the presence of immune stimulatory DNA and RNA. The production of IL‐6 is partially dependent on the interferon receptor as is IFN‐β itself. Furthermore, we have found that p204 and AIM2 are upregulated in a Type I IFN dependent fashion in vivo, in a murine model of chronic neurodegeneration. Given the propensity of inflammatory responses to cause neuronal damage, increased expression and activation of these receptors, not only during viral infection but also during sterile inflammatory responses, has the potential to exacerbate existing neuroinflammation leading to further damage and impaired neurogenesis. GLIA 2015;63:812–825
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