Invertebrate Iridescent Virus 6, a DNA Virus,Stimulates a Mammalian Innate Immune Response through RIG-I-Like Receptors

Insects are not only major vectors of mammalian viruses, but are also host to insect-restricted viruses that can potentially be transmitted to mammals. While mammalian innate immune responses to arboviruses are well studied, less is known about how mammalian cells respond to viruses that are restricted to infect only invertebrates. Here we demonstrate that IIV-6, a DNA virus of the family Iridoviridae, is able to induce a type I interferon-dependent antiviral immune response in mammalian cells. Although IIV-6 is a DNA virus, we demonstrate that the immune response activated during IIV-6 infection is mediated by the RIG-I-like receptor (RLR) pathway, and not the canonical DNA sensing pathway via cGAS/STING. We further show that RNA polymerase III is required for maximal IFN-β secretion, suggesting that viral DNA is transcribed by this enzyme into an RNA species capable of activating the RLR pathway. Finally, we demonstrate that the RLR-driven mammalian innate immune response to IIV-6 is functionally capable of protecting cells from subsequent infection with the arboviruses Vesicular Stomatitis virus and Kunjin virus. These results represent a novel example of an invertebrate DNA virus activating a canonically RNA sensing pathway in the mammalian innate immune response, which reduces viral load of ensuing arboviral infection.     

Balancing STING in antimicrobial defense and autoinflammation

Rapid detection of microbes is crucial for eliciting an effective immune response. Innate immune receptors survey the intracellular and extracellular environment for signs of a microbial infection. When they detect a pathogen-associated molecular pattern (PAMP), such as viral DNA, they alarm the cell about the ongoing infection. The central signaling hub in sensing of viral DNA is the stimulator of interferon genes (STING). Upon activation, STING induces downstream signaling events that ultimately result in the production of type I interferons (IFN I), important cytokines in antimicrobial defense, in particular towards viruses. In this review, we describe the molecular features of STING, including its upstream sensors and ligands, its sequence and structural conservation, common polymorphisms, and its localization. We further highlight how STING activation requires a careful balance: its activity is essential for antiviral defense, but unwanted activation through mutations or accidental recognition of self-derived DNA causes autoinflammatory diseases. Several mechanisms, such as post-translational modifications, ensure this balance by fine-tuning STING activation. Finally, we discuss how viruses evade detection of their genomes by either exploiting cells that lack a functional DNA sensing pathway as a niche or by interfering with STING activation through viral evasion molecules. Insight into STING’s exact mechanisms in health and disease will guide the development of novel clinical interventions for microbial infections, autoinflammatory diseases, and beyond.     

Mechanisms of Non-segmented Negative Sense RNA Viral Antagonism of Host RIG-I-Like Receptors

The pattern recognition receptors RIG-I-like receptors (RLRs) are critical molecules for cytosolic viral recognition and for subsequent activation of type I interferon production. The interferon signaling pathway plays a key role in viral detection and generating antiviral responses. Among the many pathogens, the non-segmented negative sense RNA viruses target the RLR pathway using a variety of mechanisms. Here, I review the current state of knowledge on the molecular mechanisms that allow non-segmented negative sense RNA virus recognition and antagonism of RLRs.     

TRIM30α Is a Negative-Feedback Regulator of the Intracellular DNA and DNA Virus-Triggered Response by Targeting STING

Uncontrolled immune responses to intracellular DNA have been shown to induce autoimmune diseases. Homeostasis regulation of immune responses to cytosolic DNA is critical for limiting the risk of autoimmunity and survival of the host. Here, we report that the E3 ubiquitin ligase tripartite motif protein 30α (TRIM30α) was induced by herpes simplex virus type 1 (HSV-1) infection in dendritic cells (DCs). Knockdown or genetic ablation of TRIM30α augmented the type I IFNs and interleukin-6 response to intracellular DNA and DNA viruses. Trim30α-deficient mice were more resistant to infection by DNA viruses. Biochemical analyses showed that TRIM30α interacted with the stimulator of interferon genes (STING), which is a critical regulator of the DNA-sensing response. Overexpression of TRIM30α promoted the degradation of STING via K48-linked ubiquitination at Lys275 through a proteasome-dependent pathway. These findings indicate that E3 ligase TRIM30α is an important negative-feedback regulator of innate immune responses to DNA viruses by targeting STING.     

Cell Type-Specific Subcellular Localization of Phospho-TBK1 in Response to Cytoplasmic Viral DNA

Cytoplasmic viral RNA and DNA are recognized by RIG-I-like receptors and DNA sensors that include DAI, IFI16, DDX41, and cGAS. The RNA and DNA sensors evoke innate immune responses through the IPS-1 and STING adaptors. IPS-1 and STING activate TBK1 kinase. TBK1 is phosphorylated in its activation loop, leading to IRF3/7 activation and Type I interferon (IFN) production. IPS-1 and STING localize to the mitochondria and endoplasmic reticulum, respectively, whereas it is unclear where phosphorylated TBK1 is localized in response to cytoplasmic viral DNA. Here, we investigated phospho-TBK1 (p-TBK1) subcellular localization using a p-TBK1-specific antibody. Stimulation with vertebrate DNA by transfection increased p-TBK1 levels. Interestingly, stimulation-induced p-TBK1 exhibited mitochondrial localization in HeLa and HepG2 cells and colocalized with mitochondrial IPS-1 and MFN-1. Hepatitis B virus DNA stimulation or herpes simplex virus type-1 infection also induced p-TBK1 mitochondrial localization in HeLa cells, indicating that cytoplasmic viral DNA induces p-TBK1 mitochondrial localization in HeLa cells. In contrast, p-TBK1 did not show mitochondrial localization in RAW264.7, L929, or T-23 cells, and most of p-TBK1 colocalized with STING in response to cytoplasmic DNA in those mammalian cells, indicating cell type-specific localization of p-TBK1 in response to cytoplasmic viral DNA. A previous knockout study showed that mouse IPS-1 was dispensable for Type I IFN production in response to cytoplasmic DNA. However, we found that knockdown of IPS-1 markedly reduced p-TBK1 levels in HeLa cells. Taken together, our data elucidated the cell type-specific subcellular localization of p-TBK1 and a cell type-specific role of IPS-1 in TBK1 activation in response to cytoplasmic viral DNA.     

Coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of STING-mediated signaling

Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV) NL63 and severe acute respiratory syndrome (SARS) CoV papain-like proteases (PLP) antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS). STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN). We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM) or SARS-CoV (PLpro-TM) inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction.     

Convergent evolution of escape from hepaciviral antagonism in primates

The ability to mount an interferon response on sensing viral infection is a critical component of mammalian innate immunity. Several viruses directly antagonize viral sensing pathways to block activation of the host immune response. Here, we show that recurrent viral antagonism has shaped the evolution of the host protein MAVS--a crucial component of the viral-sensing pathway in primates. From sequencing and phylogenetic analyses of MAVS from 21 simian primates, we found that MAVS has evolved under strong positive selection. We focused on how this positive selection has shaped MAVS' susceptibility to Hepatitis C virus (HCV). We functionally tested MAVS proteins from diverse primate species for their ability to resist antagonism by HCV, which uses its protease NS3/4A to cleave human MAVS. We found that MAVS from multiple primates are resistant to inhibition by the HCV protease. This resistance maps to single changes within the protease cleavage site in MAVS, which protect MAVS from getting cleaved by the HCV protease. Remarkably, most of these changes have been independently acquired at a single residue 506 that evolved under positive selection. We show that "escape" mutations lower affinity of the NS3 protease for MAVS and allow it to better restrict HCV replication. We further show that NS3 proteases from all other primate hepaciviruses, including the highly divergent GBV-A and GBV-C viruses, are functionally similar to HCV. We conclude that convergent evolution at residue 506 in multiple primates has resulted in escape from antagonism by hepaciviruses. Our study provides a model whereby insights into the ancient history of viral infections in primates can be gained using extant host and virus genes. Our analyses also provide a means by which primates might clear infections by extant hepaciviruses like HCV.     

Inhibition of alpha/beta interferon signaling by the NS4B protein of flaviviruses

Flaviviruses are insect-borne, positive-strand RNA viruses that have been disseminated worldwide. Their genome is translated into a polyprotein, which is subsequently cleaved by a combination of viral and host proteases to produce three structural proteins and seven nonstructural proteins. The nonstructural protein NS4B of dengue 2 virus partially blocks activation of STAT1 and interferon-stimulated response element (ISRE) promoters in cells stimulated with interferon (IFN). We have found that this function of NS4B is conserved in West Nile and yellow fever viruses. Deletion analysis shows that that the first 125 amino acids of dengue virus NS4B are sufficient for inhibition of alpha/beta IFN (IFN-alpha/beta) signaling. The cleavable signal peptide at the N terminus of NS4B, a peptide with a molecular weight of 2,000, is required for IFN antagonism but can be replaced by an unrelated signal peptide. Coexpression of dengue virus NS4A and NS4B together results in enhanced inhibition of ISRE promoter activation in response to IFN-alpha/beta. In contrast, expression of the precursor NS4A/B fusion protein does not cause an inhibition of IFN signaling unless this product is cleaved by the viral peptidase NS2B/NS3, indicating that proper viral polyprotein processing is required for anti-interferon function.     

Vesicular trafficking permits evasion of cGAS/STING surveillance during initial human papillomavirus infection

Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. One of these pathways, cGAS/STING, induces IRF3-dependent antiviral interferon (IFN) responses upon detection of cytosolic DNA. Virion-associated vDNA can activate cGAS/STING during initial viral entry and uncoating/trafficking, and thus cGAS/STING is an obstacle to many DNA viruses. HPV has a unique vesicular trafficking pathway compared to many other DNA viruses. As the capsid uncoats within acidic endosomal compartments, minor capsid protein L2 protrudes across vesicular membranes to facilitate transport of vDNA to the Golgi. L2/vDNA resides within the Golgi lumen until G2/M, whereupon vesicular L2/vDNA traffics along spindle microtubules, tethering to chromosomes to access daughter cell nuclei. L2/vDNA-containing vesicles likely remain intact until G1, following nuclear envelope reformation. We hypothesize that this unique vesicular trafficking protects HPV from cGAS/STING surveillance. Here, we investigate cGAS/STING responses to HPV infection. DNA transfection resulted in acute cGAS/STING activation and downstream IFN responses. In contrast, HPV infection elicited minimal cGAS/STING and IFN responses. To determine the role of vesicular trafficking in cGAS/STING evasion, we forced premature viral penetration of vesicular membranes with membrane-perturbing cationic lipids. Such treatment renders a non-infectious trafficking-defective mutant HPV infectious, yet susceptible to cGAS/STING detection. Overall, HPV evades cGAS/STING by its unique subcellular trafficking, a property that may contribute to establishment of infection.     

Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein

We present a novel mechanism by which viruses may inhibit the alpha/beta interferon (IFN-alpha/beta) cascade. The double-stranded RNA (dsRNA) binding protein NS1 of influenza virus is shown to prevent the potent antiviral interferon response by inhibiting the activation of interferon regulatory factor 3 (IRF-3), a key regulator of IFN-alpha/beta gene expression. IRF-3 activation and, as a consequence, IFN-beta mRNA induction are inhibited in wild-type (PR8) influenza virus-infected cells but not in cells infected with an isogenic virus lacking the NS1 gene (delNS1 virus). Furthermore, NS1 is shown to be a general inhibitor of the interferon signaling pathway. Inhibition of IRF-3 activation can be achieved by the expression of wild-type NS1 in trans, not only in delNS1 virus-infected cells but also in cells infected with a heterologous RNA virus (Newcastle disease virus). We propose that inhibition of IRF-3 activation by a dsRNA binding protein significantly contributes to the virulence of influenza A viruses and possibly to that of other viruses.     

Attenuation of cGAS‐STING signaling is mediated by a p62/SQSTM1‐dependent autophagy pathway activated by TBK1

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.     

Novel Bat Influenza Virus NS1 Proteins Bind Double-Stranded RNA and Antagonize Host Innate Immunity

We demonstrate that novel bat HL17NL10 and HL18NL11 influenza virus NS1 proteins are effective interferon antagonists but do not block general host gene expression. Solving the RNA-binding domain structures revealed the canonical NS1 symmetrical homodimer, and RNA binding required conserved basic residues in this domain. Interferon antagonism was strictly dependent on RNA binding, and chimeric bat influenza viruses expressing NS1s defective in this activity were highly attenuated in interferon-competent cells but not in cells unable to establish antiviral immunity.     

Discovery of IACS-8803 and IACS-8779, potent agonists of stimulator of interferon genes (STING) with robust systemic antitumor efficacy

Activation of the stimulator of interferon genes (STING) pathway by both exogenous and endogenous cytosolic DNA results in the production of interferon beta (IFN-β) and is required for the generation of cytotoxic T-cell priming against tumor antigens. In the clinical setting, pharmacological stimulation of the STING pathway has the potential to synergize with immunotherapy antibodies by boosting anti-tumor immune responses. We report the discovery of two highly potent cyclic dinucleotide STING agonists, IACS-8803 and IACS-8779, which show robust activation of the STING pathway in vitro and a superior systemic anti-tumor response in the B16 murine model of melanoma when compared to one of the clinical benchmark compounds.     

Paramyxovirus Activation and Inhibition of Innate Immune Responses

Paramyxoviruses represent a remarkably diverse family of enveloped nonsegmented negative-strand RNA viruses, some of which are the most ubiquitous disease-causing viruses of humans and animals. This review focuses on paramyxovirus activation of innate immune pathways, the mechanisms by which these RNA viruses counteract these pathways, and the innate response to paramyxovirus infection of dendritic cells (DC). Paramyxoviruses are potent activators of extracellular complement pathways, a first line of defense that viruses must face during natural infections. We discuss mechanisms by which these viruses activate and combat complement to delay neutralization. Once cells are infected, virus replication drives type I interferon (IFN) synthesis that has the potential to induce a large number of antiviral genes. Here we describe four approaches by which paramyxoviruses limit IFN induction: by limiting synthesis of IFN-inducing aberrant viral RNAs, through targeted inhibition of RNA sensors, by providing viral decoy substrates for cellular kinase complexes, and through direct blocking of the IFN promoter. In addition, paramyxoviruses have evolved diverse mechanisms to disrupt IFN signaling pathways. We describe three general mechanisms, including targeted proteolysis of signaling factors, sequestering cellular factors, and upregulation of cellular inhibitors. DC are exceptional cells with the capacity to generate adaptive immunity through the coupling of innate immune signals and T cell activation. We discuss the importance of innate responses in DC following paramyxovirus infection and their consequences for the ability to mount and maintain antiviral T cells.