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Retinoic acid–inducible gene I–like receptors (RLRs) are cytosolic RNA sensors critical for initiation of antiviral immunity. Activation of RLRs following RNA recognition leads to production of antiviral genes and IFNs for induction of broad antiviral immunity. Although the RLRs are ubiquitously expressed, much of our understanding of these molecules comes from their study in epithelial cells and fibroblasts. However, RLR activation is critical for induction of immune function and long-term protective immunity. Recent work has focused on the roles of RLRs in immune cells and their contribution to programming of effective immune responses. This new understanding of RLR function in immune cells and immune programming has led to the development of vaccines and therapeutics targeting the RLRs. This review covers recent advances in our understanding of the contribution of RLRs to immune cell function during infection and the emerging RLR-targeting strategies for induction of immunity against cancer and viral infection.

International audience; Type III interferons or IFN-λs, are composed of 4 members: IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4. IFN-λs are master protectors against mucosal viral infections due to the preferential expression of their cognate receptor, IFNLR1, to cells of epithelial lineage and to a restricted pool of immune cells. IFN-λ receptor activation induces intracellular signaling that overlaps with that of type I interferons (IFN-I) and culminates with the induction of Interferon-Stimulated-Genes (ISG) with antiviral functions. However, several studies have uncovered non-redundant functions of IFN-λs, compared to IFN-I, that go beyond viral restriction at epithelial barriers. In this topic, we highlighted several emerging aspects of IFN-λ biology, including: their ability to restrict non-viral infections, the mechanisms by which they modulate immune responses, and the enigmatic role of human IFN-λ4.

IFNs are comprised of three families of cytokines that confer protection against pathogen infection and uncontrolled cellular proliferation. The broad role IFNs play in innate and adaptive immune regulation has placed them under heavy scrutiny to position them as “friend” or “foe” across pathologies. Genetic lesions in genes involving IFN synthesis and signaling underscore the disparate outcomes of aberrant IFN signaling. Abrogation of the response leads to susceptibility to microbial infections whereas unabated IFN induction underlies a variety of inflammatory diseases and tumor immune evasion. Type I and III IFNs have overlapping roles in antiviral protection, yet the mechanisms by which they are induced and promote the expression of IFN-stimulated genes and inflammation can distinguish their biological functions. In this review, we examine the molecular factors that shape the shared and distinct roles of type I and III IFNs in immunity.

Interferons (IFN) are antiviral cytokines induced by recognition of viral nucleic acids or cellular replicative stress. In particular, the accumulation of cytosolic nucleic acids or the accumulation of DNA lesions has the potential to activate cGAS/STING-mediated IFN induction if not properly constrained. While these responses can be harnessed as tumor therapies, the excessive induction of IFNs and deleterious IFN-mediated inflammation is associated with tissue damage in non-malignant disease. We sought to identify non-canonical IFN-modulatory transcription factors (TF) by examining factors with known genetic alterations that associate with both malignant and inflammatory diseases. Here, we identify MEF2A as a negative regulator of the production of IFNs at homeostasis. The loss of MEF2A function results in the spontaneous induction of type I and III IFNs and downstream ISG expression. We demonstrate that IFN production following MEF2A depletion was dependent on DNA replication and cell cycle progression with CDK2 inhibition ablating this response. Furthermore, DNA lesions that induced DNA damage responses following MEF2A loss resulted in cGAS/STING pathway activation and genetic deletion of STING maintained homeostatic levels of type I IFN in the absence of this TF factor. Thus, our study is first to connect MEF2A with protection from maladaptive type I IFN responses due to genomic instability across various cell types. Overall, our study reveals that therapeutic modulation of MEF2A or other members of its family could be beneficial for the management of viral, malignant, and autoinflammatory diseases.

MicroRNAs (miRNAs) play an important role in fine-tuning host immune homeostasis and responses through the regulation of messenger RNA (mRNA) stability and translation. Studies have demonstrated that miRNA-mediated regulation of gene expression has a profound impact on immune cell development, function and response to invading pathogens. As we continue to examine the mechanisms by which miRNAs maintain the balance between robust protective host immune responses and dysregulated responses that promote immune pathology, careful consideration of the complexity of post-transcriptional immune regulation is needed. Distinct tissue- and stimulus-specific RNA-RNA and RNA-protein interactions can modulate the functions of a given miRNA. Thus, new challenges emerge in the identification of post-transcriptional co-regulatory modules and the genetic factors that impact miRNA function.

The initial response to viral infection is anticipatory, with host antiviral restriction factors and pathogen sensors constantly surveying the cell to rapidly mount an antiviral response through the synthesis and downstream activity of interferons. After pathogen clearance, the host's ability to resolve this antiviral response and return to homeostasis is critical. Here, we found that isoforms of the RNA-binding protein ZAP functioned as both a direct antiviral restriction factor and an interferon-resolution factor. The short isoform of ZAP bound to and mediated the degradation of several host interferon messenger RNAs, and thus acted as a negative feedback regulator of the interferon response. In contrast, the long isoform of ZAP had antiviral functions and did not regulate interferon. The two isoforms contained identical RNA-targeting domains, but differences in their intracellular localization modulated specificity for host versus viral RNA, which resulted in disparate effects on viral replication during the innate immune response.

Summary Type I and III interferons (IFNs) activate similar downstream signaling cascades, but unlike type I IFNs, type III IFNs (IFNλ) do not elicit strong inflammatory responses in vivo. Here, we examined the molecular mechanisms underlying this disparity. Type I and III IFNs displayed kinetic differences in expression of IFN-stimulated genes and proinflammatory responses, with type I IFNs preferentially stimulating expression of the transcription factor IRF1. Type III IFNs failed to induce IRF1 expression because of low IFNλ receptor abundance and insufficient STAT1 activation on epithelial cells and thus did not activate the IRF1 proinflammatory gene program. Rather, IFNλ stimulation preferentially induced factors implicated in tissue repair. Our findings suggest that IFN receptor compartmentalization and abundance confer a spatiotemporal division of labor where type III IFNs control viral spread at the site of the infection while restricting tissue damage; the transient induction of inflammatory responses by type I IFNs recruits immune effectors to promote protective immunity.

Polyomaviruses encode a large T Ag (LT), a multifunctional protein essential for the regulation of both viral and host cell gene expression and productive viral infection. Previously, we have shown that stable expression of LT protein results in upregulation of genes involved in the IFN induction and signaling pathway. In this study, we focus on the cellular signaling mechanism that leads to the induction of IFN responses by LT. Our results show that ectopic expression of SV40 LT results in the induction of IFN-stimulated genes (ISGs) in human fibroblasts and confers an antiviral state. We describe a LT-initiated DNA damage response (DDR) that activates IFN regulatory factor 1, causing IFN-β production and consequent ISG expression in human cells. This IFN-β and ISG induction is dependent on ataxia-telangiectasia mutated and Rad3-related (ATR) kinase, but independent of ATM. ATR kinase inhibition using a selective kinase inhibitor (ETP-46464) caused a decrease in IFN regulatory factor 1 stabilization and ISG expression. Furthermore, expression of a mutant LT that does not induce DDR also does not induce IFN-β and ISGs. These results show that, in the absence of viral infection, LT-initiated activation of ATR-dependent DDR is sufficient for the induction of an IFN-β–mediated innate immune response in human cells. Thus, we have uncovered a novel and critical role for ATR as a mediator of antiviral responses utilizing LT.

Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN) and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein has antiviral activity and mediates RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone-marrow-derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL, Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation.

NOD-like receptor protein 3 (NLRP3) is an intracellular sensor and a major component of the canonical inflammasome pathway, which causes inflammation and cell death when activated by two temporally discrete signals. While novel putative activators of the NLRP3 inflammasome have been identified, the mechanisms that regulate expression of NLRP3 itself are less understood. Alternative polyadenylation (AP) is a mechanism by which cells modulate expression through varying the length of the 3′ untranslated region (3′UTR). Through AP, the longer forms may provide unique regulatory elements, that allow for differential regulation of mRNA stability and translation. Here we sought to thoroughly characterize the features of the NLRP3 3′UTR and hypothesized that AP may significantly control the threshold of inflammasome activation. We examined AP in THP-1 monocytes and found that they express both long and short forms, with the long form demonstrating decreased stability relative to the short. We have attributed these differences to the presence of AU-rich elements (ARE), as ablation of AREs results in increased stability of the long NLRP3 mRNA. Additionally, we have demonstrated that a known post-transcriptional regulator of NLRP3, miR-223, preferentially regulates the long transcript as well as augments the kinetics of pyroptotic cell death. These studies demonstrate that alternative polyadenylation of the 3′UTR of NLRP3 has a marked impact on the threshold of inflammasome activation.

IFN regulatory factor (IRF) 4 is a hematopoietic cell–specific transcription factor that regulates the maturation and differentiation of immune cells. Using an inducible expression system, we found that IRF4 directly induced a specific subset of IFN-stimulated genes (ISGs) in a type I IFN–independent manner in both epithelial and B cell lines. Moreover, Kaposi sarcoma–associated herpesvirus (KSHV)–encoded viral FLICE inhibitory protein (vFLIP) enhances IRF4-mediated gene induction. Coexpression of IRF4 with vFLIP significantly increased ISG60 (IFIT3) and Cig5 (RSAD2) transcription that was dependent on the ability of vFLIP to activate NF-κB. A vFLIP mutant (A57L) defective in NF-κB activation failed to enhance IRF4-mediated ISG induction. Thus, we provide a physiologically relevant mechanism by which viral protein–mediated NF-κB activation modulates specific ISG induction by IRF4. In contrast, IRF4 also acted as a negative regulator of KSHV replication and transcription activator expression after induction of KSHV lytic reactivation in KSHV-positive primary effusion lymphoma cells. Taken together, these results suggest a dual role for IRF4 in regulating ISG induction and KSHV lytic reactivation in primary effusion lymphoma cells.

The severity of influenza virus infection is largely determined by the interplay between the virus and the host. Understanding the pathological drivers of influenza A viruses serves to rapidly characterize the host response to newly emerging influenza viruses and derive prognostic signatures of disease severity. We implemented a differential network approach to uncover novel distinctions between the global transcriptional host response to highly pathogenic and low pathogenic H1N1, H7N9, H7N7, and H5N1 influenza A infections in BALB/c mice. We identified crucial mediators of inflammatory, coagulation, and tissue repair responses that discriminate between disease outcomes. It has been demonstrated that these responses are largely impacted by the immune cell influx into the site of infection. We examined the contribution of resident and lung-infiltrating immune cell quantity and activation state to disease phenotypes by implementing digital cell quantification (DCQ), which allowed us to model the in vivo dynamics of immune cells across viral strains based on time and strain-dependent infected lung transcriptional profiles. Through linear regression modeling, we revealed that distinct monocyte, granulocyte, DC, and T cell populations predict disease morbidity. Additionally, we demonstrated that both the kinetics of CD8+ T cell infiltration and the extent of their activation state, serve as a key predictor of immunopathological consequences of influenza infection. Overall, we provide a multidimensional analysis that delineates how innate immune responses relate to aberrant tissue repair responses and drive subsequent host adaptive immune response that control the severity of influenza virus disease in the murine model.

Virus infection is sensed in the cytoplasm by RIG-I, which requires RNA and poly-ubiquitin binding to induce type I interferon (IFN), and activate cellular innate immunity. We show that unlike mouse, the human IFN-inducible oligoadenylate synthetases-like (OASL) protein has antiviral activity and mediates RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduces RIG-I signaling and enhances virus replication in human cells. Conversely, OASL expression suppresses replication of a number of viruses in a RIG-I-dependent manner and enhances RIG-I-mediated IFN induction. OASL interacts and colocalizes with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhances RIG-I signaling. Importantly, inactive RIG-I mutants defective in ubiquitination and polyubiquitin binding still bind to OASL, and their signaling activity is rescued by OASL. Our findings show a novel mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation.

Oligoadenylate Synthetases (OAS) are interferon (IFN) inducible enzymes, which contribute to the host anti-viral activity by inducing RNA degradation via classical OAS-RNaseL pathway. However, the OAS-like protein (OASL), which is strongly induced following virus infection, does not have enzymatic activity to synthesize 2’-5’ oligoadenylates. Therefore, the mechanism of the anti-viral activity of OASL has remained elusive. Here we describe the basis of anti-viral activity of OASL via its ability to modulate type I IFN induction through RIG-I pathway. OASL expressing cells show a strong anti-viral activity against a number of RNA and DNA virus infections in a RIG-I dependent manner. Expression of OASL enhances RIG-I mediated IFN induction. We show that OASL interacts with cytoplasmic viral RNA sensor RIG-I and enhance its activity through its C-terminal ubiquitin like domain. Poly ubiquitin binding to RIG-I is necessary and essential for RIG-I mediated induction of IFN. We demonstrate that following induction of OASL protein it provides the necessary poly Ub binding function of RIG-I. Furthermore, inactive RIG-I mutants, which are defective in ubiquitination and poly Ub binding can be rescued in presence of OASL. We show that OASL specifically enhances RIG-I mediated IFN induction without affecting MDA5, STING activation. Our findings suggest a novel mechanism by which OASL contributes to host anti-viral response.