Jiaxi Wu

Cyclic GMP-AMP Synthase Is an Innate Immune Sensor of HIV and Other Retroviruses

HIV Detection Is a (c)GAS Despite it being one of the most highly studied viruses, there are still many unknowns when it comes to HIV—including how it triggers the innate immune response. Gao et al. (p. 903, published 8 August) now demonstrate that the DNA sensor cyclic GMP-AMP synthase (cGAS) detects HIV infection. Reverse-transcribed HIV DNA triggers cGAS and downstream activation of antiviral immunity. Detection of HIV, as well as the retroviruses simian immunodeficiency virus and murine leukemia virus, was abrogated in mouse and human cells deficient in cGAS—suggesting that cGAS may be a critical activator of innate immunity in response to retroviral infection. Cell culture experiments suggest that detection of retroviral DNA activates cellular defense systems. Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-β induction by the virus, suggesting that the reverse-transcribed HIV DNA triggers the innate immune response. Knockout or knockdown of cGAS in mouse or human cell lines blocked cytokine induction by HIV, murine leukemia virus, and simian immunodeficiency virus. These results indicate that cGAS is an innate immune sensor of HIV and other retroviruses.

Pivotal Roles of cGAS-cGAMP Signaling in Antiviral Defense and Immune Adjuvant Effects

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. The cytosolic DNA sensor cyclic guanosine monophosphate–adenosine monophosphate synthase is essential for antiviral immunity in vivo. Invasion of microbial DNA into the cytoplasm of animal cells triggers a cascade of host immune reactions that help clear the infection; however, self DNA in the cytoplasm can cause autoimmune diseases. Biochemical approaches led to the identification of cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) as a cytosolic DNA sensor that triggers innate immune responses. Here, we show that cells from cGAS-deficient (cGas−/−) mice, including fibroblasts, macrophages, and dendritic cells, failed to produce type I interferons and other cytokines in response to DNA transfection or DNA virus infection. cGas−/− mice were more susceptible to lethal infection with herpes simplex virus 1 (HSV1) than wild-type mice. We also show that cGAMP is an adjuvant that boosts antigen-specific T cell activation and antibody production in mice.

Innate Immune Sensing and Signaling of Cytosolic Nucleic Acids

The innate immune system utilizes pattern-recognition receptors (PRRs) to detect the invasion of pathogens and initiate host antimicrobial responses such as the production of type I interferons and proinflammatory cytokines. Nucleic acids, which are essential genetic information carriers for all living organisms including viral, bacterial, and eukaryotic pathogens, are major structures detected by the innate immune system. However, inappropriate detection of self nucleic acids can result in autoimmune diseases. PRRs that recognize nucleic acids in cells include several endosomal members of the Toll-like receptor family and several cytosolic sensors for DNA and RNA. Here, we review the recent advances in understanding the mechanism of nucleic acid sensing and signaling in the cytosol of mammalian cells as well as the emerging role of cytosolic nucleic acids in autoimmunity.

Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation

Innate immune receptor signaling, united Innate immune receptors such as RIG-I, cGAS, and Toll-like receptors bind microbial fragments and alert the immune system to an infection. Each receptor type signals through a different adapter protein. These signals activate the protein kinase TBK1 and the transcription factor IRF3, which tells cells to secrete interferon proteins (IFNs) important for host defense. Liu et al. now report a common signaling mechanism used by all three types of innate immune receptor-adaptor protein pairs to activate IRF3 and generate IFNs. This is important because cells must regulate their IFN production carefully to avoid inflammation and autoimmunity. Science, this issue 10.1126/science.aaa2630 Diverse innate immune receptors use a common signaling mechanism to activate type I interferons. INTRODUCTION Sensing of pathogenic microbes and tissue damage by the innate immune system triggers immune cells to secrete cytokines that promote host defense. Viral RNA, cytosolic DNA, and the bacterial cell wall component lipopolysaccharide activate signaling cascades through a number of pattern recognition receptor (PRR)–adaptor protein pairs, including RIG-I–MAVS, cGAS-STING, and TLR3/4-TRIF (TLR3/4, Toll-like receptors 3 and 4). Activation of these signaling modules results in the production of type I interferons (IFNs), a family of cytokines that are essential for host protection. The adaptor proteins MAVS, STING, and TRIF each activate the downstream protein kinase TBK1, which then phosphorylates the transcription factor interferon regulatory factor 3 (IRF3), which drives type I IFN production. Although much progress has been made in our understanding of PRR and adaptor protein activation, the mechanism by which the adaptor proteins activate TBK1 and IRF3 remains unclear. RATIONALE Other signaling pathways besides the RIG-I–MAVS, cGAS-STING, and TLR3/4-TRIF pathways activate TBK1. However, IRF3 phosphorylation by TBK1 is observed only in the IFN-producing pathways that use MAVS, STING, or TRIF as the adaptor protein. The discrepant activation of TBK1 and IRF3 implies the existence of a kinase-substrate specification mechanism exclusive to the IFN-producing pathways. Specification of TBK1-mediated IRF3 activation is essential for the tight regulation of IFN production, which would otherwise lead to autoimmune diseases. RESULTS Using biochemical and mouse cell– and human cell–based assays, we found that both MAVS and STING interacted with IRF3 in a phosphorylation-dependent manner. We show that both MAVS and STING are phosphorylated in response to stimulation at their respective C-terminal consensus motif, pLxIS (p, hydrophilic residue; x, any residue; S, phosphorylation site). This phosphorylation event then recruits IRF3 to the active adaptor protein and is essential for IRF3 activation. Point mutations that impair the phosphorylation of MAVS or STING at their consensus motif abrogated IRF3 binding and subsequent IFN induction. We found that MAVS is phosphorylated by the kinases TBK1 and IKK, whereas STING is phosphorylated by TBK1. Phosphorylated MAVS and STING subsequently bind to conserved, positively charged surfaces of IRF3, thereby recruiting IRF3 for its phosphorylation and activation by TBK1. Point mutations at IRF3’s positively charged surfaces abrogated IRF3 binding to MAVS and STING and subsequent IRF3 phosphorylation and activation. We further show that TRIF-mediated activation of IRF3 depends on TRIF phosphorylation at the pLxIS motif commonly found in MAVS, STING, and IRF3. These results reveal that phosphorylation of innate immune adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate type I IFN production. CONCLUSION We uncovered a general mechanism of IRF3 activation by the innate immune adaptor proteins MAVS, STING, and TRIF, which functions in three distinct pattern recognition pathways. Following its activation, each adaptor protein recruits and activates downstream kinase TBK1, which phosphorylates the cognate upstream adaptor protein at a consensus motif. Phosphorylated MAVS, STING, or TRIF in turn recruits IRF3 through its conserved, positively charged phospho-binding domain, allowing IRF3 phosphorylation by TBK1. Phosphorylated IRF3 subsequently dissociates from the adaptor protein and dimerizes though the same phospho-binding domain before translocating into the nucleus to induce IFN. These results elucidate how IRF3 activation and IFN production are tightly controlled and explain why TBK1 is necessary but not sufficient to phosphorylate IRF3: Phosphorylation of IRF3 by TBK1 occurs only with the assistance of an adaptor protein such as MAVS, STING, or TRIF, which also must be phosphorylated. Phosphorylation of innate immune adaptor proteins licenses IRF3 activation. MAVS, STING, and TRIF—which are activated by viral RNA, cytosolic DNA, and bacterial lipopolysaccharide (LPS), respectively—activate the kinases IKK and TBK1. These kinases then phosphorylate the adaptor proteins, which in turn recruit IRF3, thereby licensing IRF3 for phosphorylation (P) by TBK1. Phosphorylated IRF3 dissociates from the adaptor proteins, dimerizes, and then enters the nucleus to induce IFNs. During virus infection, the adaptor proteins MAVS and STING transduce signals from the cytosolic nucleic acid sensors RIG-I and cGAS, respectively, to induce type I interferons (IFNs) and other antiviral molecules. Here we show that MAVS and STING harbor two conserved serine and threonine clusters that are phosphorylated by the kinases IKK and/or TBK1 in response to stimulation. Phosphorylated MAVS and STING then bind to a positively charged surface of interferon regulatory factor 3 (IRF3) and thereby recruit IRF3 for its phosphorylation and activation by TBK1. We further show that TRIF, an adaptor protein in Toll-like receptor signaling, activates IRF3 through a similar phosphorylation-dependent mechanism. These results reveal that phosphorylation of innate adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate the type I IFN pathway.

MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades

RNA virus infections are detected by the RIG-I family of receptors, which induce type-I interferons through the mitochondrial protein MAVS. MAVS forms large prion-like polymers that activate the cytosolic kinases IKK and TBK1, which in turn activate NF-κB and IRF3, respectively, to induce interferons. Here we show that MAVS polymers recruit several TRAF proteins, including TRAF2, TRAF5, and TRAF6, through distinct TRAF-binding motifs. Mutations of these motifs that disrupted MAVS binding to TRAFs abrogated its ability to activate IRF3. IRF3 activation was also abolished in cells lacking TRAF2, 5, and 6. These TRAF proteins promoted ubiquitination reactions that recruited NEMO to the MAVS signaling complex, leading to the activation of IKK and TBK1. These results delineate the mechanism of MAVS signaling and reveal that TRAF2, 5, and 6, which are normally associated with NF-κB activation, also play a crucial role in IRF3 activation in antiviral immune responses. DOI: http://dx.doi.org/10.7554/eLife.00785.001

The Cytosolic DNA Sensor cGAS Forms an Oligomeric Complex with DNA and Undergoes Switch-like Conformational Changes in the Activation Loop

The presence of DNA in the cytoplasm is a danger signal that triggers immune and inflammatory responses. Cytosolic DNA binds to and activates cyclic GMP-AMP (cGAMP) synthase (cGAS), which produces the second messenger cGAMP. cGAMP binds to the adaptor protein STING and activates a signaling cascade that leads to the production of type I interferons and other cytokines. Here, we report the crystal structures of human cGAS in its apo form, representing its autoinhibited conformation as well as in its cGAMP- and sulfate-bound forms. These structures reveal switch-like conformational changes of an activation loop that result in the rearrangement of the catalytic site. The structure of DNA-bound cGAS reveals a complex composed of dimeric cGAS bound to two molecules of DNA. Functional analyses of cGAS mutants demonstrate that both the protein-protein interface and the two DNA binding surfaces are critical for cGAS activation. These results provide insights into the mechanism of DNA sensing by cGAS.

An Argonaute phosphorylation cycle promotes microRNA-mediated silencing

MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824–S834), followed by rapid dephosphorylation by the ANKRD52–PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.

Molecular basis for the specific recognition of the metazoan cyclic GMP-AMP by the innate immune adaptor protein STING

Significance The presence of cytosolic DNA in mammalian cells signifies microbial invasions and triggers the DNA sensor protein cGAS to produce the second messenger molecule 2′3′-cGAMP, which elicits innate immune responses by binding to and activating the homodimerized adaptor protein STING. Here we show that the high affinity of the asymmetric ligand 2′3′-cGAMP to the symmetric dimer of STING originates from its unique mixed phosphodiester linkages. 2′3′-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low energy costs in changing into the active conformation. Whereas biological structural studies have focused on analyses of protein conformations, our results demonstrate that analyses of free-ligand conformations can be equally important in understanding protein–ligand interactions. Cyclic GMP-AMP containing a unique combination of mixed phosphodiester linkages (2′3′-cGAMP) is an endogenous second messenger molecule that activates the type-I IFN pathway upon binding to the homodimer of the adaptor protein STING on the surface of endoplasmic reticulum membrane. However, the preferential binding of the asymmetric ligand 2′3′-cGAMP to the symmetric dimer of STING represents a physicochemical enigma. Here we show that 2′3′-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low entropy and enthalpy costs in converting into the active conformation. Our results demonstrate that analyses of free-ligand conformations can be as important as analyses of protein conformations in understanding protein–ligand interactions.

BHLHE40, a third transcription factor required for insulin induction of SREBP-1c mRNA in rodent liver

In obesity, elevated insulin causes fatty liver by activating the gene encoding SREBP-1c, a transcription factor that enhances fatty acid synthesis. Two transcription factors, LXRα and C/EBPβ, are necessary but not sufficient for insulin induction of hepatic SREBP-1c mRNA. Here, we show that a third transcription factor, BHLHE40, is required. Immunoprecipitation revealed that BHLHE40 binds to C/EBPβ and LXRα in livers of rats that had fasted and then refed. Hepatic BHLHE40 mRNA rises rapidly when fasted rats are refed and when rat hepatocytes are incubated with insulin. Preventing this rise by gene knockout in mice or siRNAs in hepatocytes reduces the insulin-induced rise in SREBP-1c mRNA. Although BHLHE40 is necessary for insulin induction of SREBP-1c, it is not sufficient as demonstrated by failure of lentiviral BHLHE40 overexpression to increase hepatocyte SREBP-1c mRNA in the absence of insulin. Thus, an additional event is required for insulin to increase SREBP-1c mRNA.