Cytosolic DNA arising from intracellular pathogens triggers a powerful innate immune response. It is sensed by cyclic GMP-AMP synthase (cGAS), which elicits the production of type I interferons by generating the second messenger 2'3'-cyclic-GMP-AMP (cGAMP). Endogenous nuclear or mitochondrial DNA can also be sensed by cGAS under certain conditions, resulting in sterile inflammation. The cGAS dimer binds two DNA ligands shorter than 20 base pairs side-by-side, but 20-base-pair DNA fails to activate cGAS in vivo and is a poor activator in vitro. Here we show that cGAS is activated in a strongly DNA length-dependent manner both in vitro and in human cells. We also show that cGAS dimers form ladder-like networks with DNA, leading to cooperative sensing of DNA length: assembly of the pioneering cGAS dimer between two DNA molecules is ineffective; but, once formed, it prearranges the flanking DNA to promote binding of subsequent cGAS dimers. Remarkably, bacterial and mitochondrial nucleoid proteins HU and mitochondrial transcription factor A (TFAM), as well as high-mobility group box 1 protein (HMGB1), can strongly stimulate long DNA sensing by cGAS. U-turns and bends in DNA induced by these proteins pre-structure DNA to nucleate cGAS dimers. Our results suggest a nucleation-cooperativity-based mechanism for sensitive detection of mitochondrial DNA and pathogen genomes, and identify HMGB/TFAM proteins as DNA-structuring host factors. They provide an explanation for the peculiar cGAS dimer structure and suggest that cGAS preferentially binds incomplete nucleoid-like structures or bent DNA.

cGAS senses long and HMGB/TFAM-bound U-turn DNA by forming protein–DNA ladders

Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62-deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy-associated vesicles. Thus, DNA sensing induces the cGAS-STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.

https://www.embopress.org/doi/pdf/10.15252/embj.201797858

Attenuation of cGAS-STING Signaling Is Mediated by a p62/SQSTM1-dependent Autophagy Pathway Activated by TBK1

Cytosolic DNA stimulates innate immune responses, including type I interferons (IFN), which have antiviral and immunomodulatory activities. Cyclic GMP-AMP synthase (cGAS) recognizes cytoplasmic DNA and signals via STING to induce IFN production. Despite the importance of DNA in innate immunity, the nature of the DNA that stimulates IFN production is not well described. Using low DNA concentrations, we show that dsDNA induces IFN in a length-dependent manner. This is observed over a wide length-span of DNA, ranging from the minimal stimulatory length to several kilobases, and is fully dependent on cGAS irrespective of DNA length. Importantly, in vitro studies reveal that long DNA activates recombinant human cGAS more efficiently than short DNA, showing that length-dependent DNA recognition is an intrinsic property of cGAS independent of accessory proteins. Collectively, this work identifies long DNA as the molecular entity stimulating the cGAS pathway upon cytosolic DNA challenge such as viral infections.

/pdf/cgas-is-activated-by-dna-in-a-length-dependent-manner-2qpxykxn27.pdf

cGAS is activated by DNA in a length-dependent manner.

The induction of type I interferons through the transcription factor interferon regulatory factor 3 (IRF3) is considered a major outcome of stimulator of interferon genes (STING) activation that drives immune responses against DNA viruses and tumors. However, STING activation can also trigger other downstream pathways such as nuclear factor κB (NF-κB) signaling and autophagy, and the roles of interferon (IFN)-independent functions of STING in infectious diseases or cancer are not well understood. Here, we generated a STING mouse strain with a mutation (S365A) that disrupts IRF3 binding and therefore type I interferon induction but not NF-κB activation or autophagy induction. We also generated STING mice with mutations that disrupt the recruitment of TANK-binding kinase 1 (TBK1), which is important for both IRF3 and NF-κB activation but not autophagy induction (L373A or ∆CTT, which lacks the C-terminal tail). The STING-S365A mutant mice, but not L373A or ∆CTT mice, were still resistant to herpes simplex virus 1 (HSV-1) infections and mounted an antitumor response after cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) treatment despite the absence of STING-induced interferons. These results demonstrate that STING can function independently of type I interferons and autophagy, and that TBK1 recruitment to STING is essential for antiviral and antitumor immunity.

https://www.pnas.org/content/pnas/118/14/e2100225118.full.pdf

TBK1 recruitment to STING activates both IRF3 and NF-κB that mediate immune defense against tumors and viral infections.

Double-stranded DNA (dsDNA) sensor cyclic-GMP-AMP synthase (cGAS) along with the downstream stimulator of interferon genes (STING) acting as essential immune-surveillance mediators have become hot topics of research. The intrinsic function of the cGAS-STING pathway facilitates type-I interferon (IFN) inflammatory signaling responses and other cellular processes such as autophagy, cell survival, senescence. cGAS-STING pathway interplays with other innate immune pathways, by which it participates in regulating infection, inflammatory disease, and cancer. The therapeutic approaches targeting this pathway show promise for future translation into clinical applications. Here, we present a review of the important previous works and recent advances regarding the cGAS-STING pathway, and provide a comprehensive understanding of the modulatory pattern of the cGAS-STING pathway under multifarious pathologic states.

/pdf/research-advances-in-how-the-cgas-sting-pathway-controls-the-3ea3v3cmvc.pdf

Research Advances in How the cGAS-STING Pathway Controls the Cellular Inflammatory Response.

Herpes simplex encephalitis (HSE) is the most common form of acute viral encephalitis in industrialized countries. Type I interferon (IFN) is important for control of herpes simplex virus (HSV-1) in the central nervous system (CNS). Here we show that microglia are the main source of HSV-induced type I IFN expression in CNS cells and these cytokines are induced in a cGAS-STING-dependent manner. Consistently, mice defective in cGAS or STING are highly susceptible to acute HSE. Although STING is redundant for cell-autonomous antiviral resistance in astrocytes and neurons, viral replication is strongly increased in neurons in STING-deficient mice. Interestingly, HSV-infected microglia confer STING-dependent antiviral activities in neurons and prime type I IFN production in astrocytes through the TLR3 pathway. Thus, sensing of HSV-1 infection in the CNS by microglia through the cGAS-STING pathway orchestrates an antiviral program that includes type I IFNs and immune-priming of other cell types.

https://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=4000&context=oapubs

Sensing of HSV-1 by the cGAS-STING pathway in microglia orchestrates antiviral defence in the CNS

NKT cell activation by local α-galactosylceramide administration decreases susceptibility to HSV-2 infection

The innate immune system constitutes the first line of defense against virus infections. This arm of the immune system uses germ line–encoded pattern recognition receptors to detect molecules which are either specific for microorganisms or present in aberrant locations during infection. Collectively, these immune-stimulating viral molecules are termed pathogen-associated molecular patterns. Virus-derived nucleic acids, including double-stranded RNA or DNA, are highly immunostimulatory, and are detected by Toll-like receptors, retinoic acid–inducible gene-I–like receptors, and cytosolic DNA sensors. Following recognition, signals are transduced to induce antiviral, inflammatory, and death programmes. Most notably, type I interferon (IFN) is a potent antiviral cytokine, and defects in production or responsiveness to IFN is associated with susceptibility to viral infections.

Henriette Winther

Papers

Sensing of HSV-1 by the cGAS-STING pathway in microglia orchestrates antiviral defence in the CNS

NKT cell activation by local α-galactosylceramide administration decreases susceptibility to HSV-2 infection

Sensors of Viral Infection