Crosstalk between Cytoplasmic RIG-I and STING Sensing Pathways

Abstract

Detection of evolutionarily conserved molecules on microbial pathogens by host immune sensors represents the initial trigger of the immune response against infection. Cytosolic receptors sense viral and intracellular bacterial genomes, as well as nucleic acids produced during replication. Once activated, these sensors trigger multiple signaling cascades, converging on the production of type I interferons and proinflammatory cytokines. Although distinct classes of receptors are responsible for the RNA and DNA sensing, the downstream signaling components are physically and functionally interconnected. This review highlights the importance of the crosstalk between retinoic acid inducible gene-I (RIG-I)-mitochondrial antiviral-signaling protein (MAVS) RNA sensing and the cyclic GMP-AMP synthase (cGAS)- stimulator of interferon genes (STING) DNA sensing pathways in potentiating efficient antiviral responses. The potential of cGAS-STING manipulation as a component of cancer immunotherapy is also reviewed.

Figure 1. Schematic Representation of the Intracellular Viral Sensing Pathways

DNA and RNA viruses release their genomes in the cytoplasm, where innate sensors for nucleic acids reside. Upon ss/dsRNA binding, RIG-I engages the adaptor protein MAVS on the mitochondria outer membrane. The cGAS receptor in contrast recognizes dsDNA and the RNA:DNA hybrids generated during retroviral replication, and catalyzes the synthesis of cGAMP, which is the paramount agonist of the adaptor protein STING. Another sensor, IFI16 can recruit STING in response to cytoplasmic DNA, through a molecular mechanism yet to be described. Both STING and MAVS stimulate downstream signaling cascades that involve multiple kinases and finally lead to IRF3 phosphorylation and nuclear translocation. The primary consequence of these virus sensing pathways is the induction of type I IFN and IFN stimulated genes. Abbreviations used: ss/dsRNA, single-stranded/double-stranded RNA, vRNA/DNA, viral RNA/DNA; RIG-I, retinoic acid inducible gene-I; MAVS, mitochondrial antiviral-signaling protein; cGAS, cyclic GMP-AMP synthase; cGAMP, 2’3’ guanosine-adenosine monophosphate; STING, stimulator of interferon genes; TANK, TRAF-associated NFκB activator; TBK1, TANK binding kinase 1; IKK, IκB kinase; IFI16, Interferon Gamma Inducible Protein 16; IRF3, interferon regulatory factor 3.

Figure 2. STING is a RIG-I Inducible Gene

In cells infected by JEV, SeV or treated with 5’pppRNA, RIG-I signaling is strongly activated; through the NF-κB and IRF3 cascades this pathway induces the production of IFNα/β and proinflammatory cytokines such as TNFα through an autocrine/paracrine manner that synergistically upregulates STING expression. By a positive feedback mechanism, induction of STING contributes to a sustained inflammatory response that protects treated cells from infection, including DNA virus infection. Abbreviations used: JEV, Japanese encephalitis virus; SeV, Sendai virus; VSV, vesicular stomatitis virus; HCV hepatitis C virus; 5’pppRNA, 5’-triphosphorylated RNA; TRAF, TNF receptor associated factor; MAVS, mitochondrial antiviral-signaling protein; RIG-I, Retinoic acid inducible gene-I; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; IκB, inhibitor of kappa B; IKK, IκB kinase; IRF, interferon regulatory factor; IFNα/β, Interferon α/β; IFNAR: IFNα receptor; JAK, Janus kinase; TYK: tyrosine kinase; STAT, signal transducer and activator of transcription; ISGF3, IFN-stimulated gene factor 3; STING, stimulator of interferon genes; HSV-1, Herpes simplex virus type 1.

Figure 3. Cross-talk between the Viral RNA and DNA Sensing Pathways

(A) AT-rich dsDNA from bacteria (Legionella) or DNA viruses (EBV, HSV-1) can be used as a template for RNA polymerase (Pol) III-driven synthesis of dsRNA bearing a 5’ triphosphate end group. Newly synthesized dsRNA binds RIG-I, activates MAVS and induces IFNβ production through TBK1-induced IRF3 phosporylation. (B) STING interacts with RIG-I and MAVS to facilitate the triggering of the antiviral response in a complex that is stabilized upon RNA virus infection. (C) Enveloped RNA viruses such as influenza A activate the STING-IFN axis independently of cGAS through a membrane fusion process. (D) The RNA genome of retroviruses is reverse transcribed in a multistep reaction that generates DNA:RNA duplexes and ssDNA as intermediates, and dsDNA molecules as final products. The cGAS-STING axis is stimulated by retroviral replicative intermediates to counteract infection and limit proviral integration via induction of ISG. Abbreviations used: HSV, herpes simplex virus; EBV, Epstein-Barr virus; SeV, Sendai virus; VSV, vesicular stomatitis virus; JEV, Japanese encephalitis virus; RIG-I, retinoic acid inducible gene-I; MAVS, mitochondrial antiviral-signaling protein; cGAS, cyclic GMP-AMP synthase; cGAMP, 2’3’ guanosine-adenosine monophosphate; STING, stimulator of interferon genes; TBK1, TANK binding kinase 1; IKK, IκB kinase; IRF3, interferon regulatory factor 3; STAT, signal transducer and activator of transcription; ISGs, interferon stimulated genes.

Figure 4. A Model of RIG-I Mediated Stimulation of Adaptive Immune Responses

1. RIG-I agonist activates cGAS-STING in DC (dendritic cells). Stimulation of the RIG-I cytosolic sensor by 5’pppRNA engages the MAVS adapter, resulting in release of reactive oxygen species (ROS) and mitochondrial DNA release that in turn stimulates cGAS-dependent production of cGAMP, activation of the STING sensor on the endoplasmic reticulum, downstream activation of IRF-3 and production of type I interferon (IFN). 2. Activated RIG-I and STING lead to type I IFN-dependent DC activation. 5’pppRNA in combination with type I IFN induces DC maturation, based on enhanced surface marker expression and increased antigen presenting capacity. 3. Activated DC drive adaptive immune responses. Activated DC engage CD4 + T cells and stimulate a Th1 skewed T cell response that includes IFNγ release, maturation of B lymphocytes to antibody producing plasma cells and the production of antibodies directed against viral or tumor antigens (14,15). Abbreviations used: RIG-I, retinoic acid inducible gene-I; MAVS, mitochondrial antiviral-signaling protein; IFN, interferon; cGAS, cyclic GMP-AMP synthase; cGAMP, 2’3’ guanosine-adenosine monophosphate; STING, stimulator of interferon genes