Distinct and Orchestrated Functions of RNA Sensors in Innate Immunity.

Faithful maintenance of immune homeostasis relies on the capacity of the cellular immune surveillance machinery to recognize "nonself", such as the presence of pathogenic RNA. Several families of pattern-recognition receptors exist that detect immunostimulatory RNA and then induce cytokine-mediated antiviral and proinflammatory responses. Here, we review the distinct features of bona fide RNA sensors, Toll-like receptors and retinoic-acid inducible gene-I (RIG-I)-like receptors in particular, with a focus on their functional specificity imposed by cell-type-dependent expression, subcellular localization, and ligand preference. Furthermore, we highlight recent advances on the roles of nucleotide-binding oligomerization domain (NOD)-like receptors and DEAD-box or DEAH-box RNA helicases in an orchestrated RNA-sensing network and also discuss the relevance of RNA sensor polymorphisms in human disease.

Stem-loop recognition by DDX17 facilitates miRNA processing and antiviral defense.

DEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening, we identify an evolutionarily conserved and interferon-independent role for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhanced RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using crosslinking immunoprecipitation high-throughput sequencing (CLIP-seq), we show that DDX17 binds the stem loops of host pri-miRNA to facilitate their processing and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous microRNA (miRNA) biogenesis and in the cytoplasm for surveillance against structured non-self-elements.

A phosphomimetic-based mechanism of dengue virus to antagonize innate immunity.

14-3-3 proteins regulate biological processes by binding to phosphorylated serine or phosphorylated threonine motifs of cellular proteins. Among the 14-3-3 proteins, 14-3-3ɛ serves a crucial function in antiviral immunity by mediating the cytosol-to-mitochondrial membrane translocation of the pathogen sensor RIG-I. Here we found that the NS3 protein of dengue virus (DV) bound to 14-3-3ɛ and prevented translocation of RIG-I to the adaptor MAVS and thereby blocked antiviral signaling. Intriguingly, a highly conserved phosphomimetic RxEP motif in NS3 was essential for the binding of 14-3-3ɛ. A recombinant mutant DV deficient in binding to 14-3-3ɛ showed impairment in antagonism of RIG-I and elicited a markedly augmented innate immune response and enhanced T cell activation. Our work reveals a novel phosphomimetic-based mechanism for viral antagonism of 14-3-3-mediated immunity, which might guide the rational design of therapeutics.

Drosha controls dendritic cell development by cleaving messenger RNAs encoding inhibitors of myelopoiesis.

To investigate if the microRNA (miRNA) pathway is required for dendritic cell (DC) development, we assessed the effect of ablating Drosha and Dicer, the two enzymes central to miRNA biogenesis. We found that while Dicer deficiency had some effect, Drosha deficiency completely halted DC development and halted myelopoiesis more generally. This indicated that while the miRNA pathway did have a role, it was a non-miRNA function of Drosha that was particularly critical. Drosha repressed the expression of two mRNAs encoding inhibitors of myelopoiesis in early hematopoietic progenitors. We found that Drosha directly cleaved stem-loop structure within these mRNAs and that this mRNA degradation was necessary for myelopoiesis. We have therefore identified a mechanism that regulates the development of DCs and other myeloid cells.

The RNA helicase DHX35 functions as a co-sensor for RIG-I-mediated innate immunity.

RNA helicases are involved in the innate immune response against pathogens, including bacteria and viruses; however, their mechanism in the human airway epithelial cells is still not fully understood. Here, we demonstrated that DEAH (Asp-Glu-Ala-His) box polypeptide 35 (DHX35), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family, boosts antiviral innate immunity in human airway epithelial cells. DHX35 knockdown attenuated the production of interferon-ß (IFN-ß), IL6, and CXCL10, whereas DHX35 overexpression increased their production. Upon stimulation, DHX35 was constitutively expressed, but it translocated from the nucleus into the cytosol, where it recognized cytosolic poly(I:C) and poly(dA:dT) via its HELICc domain. Mitochondrial antiviral signaling protein (MAVS) acted as an adaptor for DHX35 and interacted with the HELICc domain of DHX35 using amino acids 360-510. Interestingly, DHX35 interacted with retinoic acid-inducible gene 1 (RIG-I), enhanced the binding affinity of RIG-I with poly(I:C) and poly(dA:dT), and formed a signalsome with MAVS to activate interferon regulatory factor 3 (IRF3), NF-κB-p65, and MAPK signaling pathways. These results indicate that DHX35 not only acted as a cytosolic nucleic acid sensor but also synergized with RIG-I to enhance antiviral immunity in human airway epithelial cells. Our results demonstrate a novel molecular mechanism for DHX35 in RIG-I-mediated innate immunity and provide a novel candidate for drug and vaccine design to control viral infections in the human airway.

The SKIV2L RNA exosome limits activation of the RIG-I-like receptors.

Sensors of the innate immune system that detect intracellular nucleic acids must be regulated to prevent inappropriate activation by endogenous DNA and RNA. The exonuclease Trex1 regulates the DNA-sensing pathway by metabolizing potential DNA ligands that trigger it. However, an analogous mechanism for regulating the RIG-I-like receptors (RLRs) that detect RNA remains unknown. We found here that the SKIV2L RNA exosome potently limited the activation of RLRs. The unfolded protein response (UPR), which generated endogenous RLR ligands through the cleavage of cellular RNA by the endonuclease IRE-1, triggered the production of type I interferons in cells depleted of SKIV2L. Humans with deficiency in SKIV2L had a type I interferon signature in their peripheral blood. Our findings reveal a mechanism for the intracellular metabolism of immunostimulatory RNA, with implications for specific autoimmune disorders.

GEF-H1 controls microtubule-dependent sensing of nucleic acids for antiviral host defenses.

Detailed understanding of the signaling intermediates that confer the sensing of intracellular viral nucleic acids for induction of type I interferons is critical for strategies to curtail viral mechanisms that impede innate immune defenses. Here we show that the activation of the microtubule-associated guanine nucleotide exchange factor GEF-H1, encoded by Arhgef2, is essential for sensing of foreign RNA by RIG-I-like receptors. Activation of GEF-H1 controls RIG-I-dependent and Mda5-dependent phosphorylation of IRF3 and induction of IFN-ß expression in macrophages. Generation of Arhgef2(-/-) mice revealed a pronounced signaling defect that prevented antiviral host responses to encephalomyocarditis virus and influenza A virus. Microtubule networks sequester GEF-H1 that upon activation is released to enable antiviral signaling by intracellular nucleic acid detection pathways.

Diverse intracellular pathogens activate type III interferon expression from peroxisomes.

Type I interferon responses are considered the primary means by which viral infections are controlled in mammals. Despite this view, several pathogens activate antiviral responses in the absence of type I interferons. The mechanisms controlling type I interferon-independent responses are undefined. We found that RIG-I like receptors (RLRs) induce type III interferon expression in a variety of human cell types, and identified factors that differentially regulate expression of type I and type III interferons. We identified peroxisomes as a primary site of initiation of type III interferon expression, and revealed that the process of intestinal epithelial cell differentiation upregulates peroxisome biogenesis and promotes robust type III interferon responses in human cells. These findings highlight the importance of different intracellular organelles in specific innate immune responses.

RIG-I detects viral genomic RNA during negative-strand RNA virus infection.

RIG-I is a key mediator of antiviral immunity, able to couple detection of infection by RNA viruses to the induction of interferons. Natural RIG-I stimulatory RNAs have variously been proposed to correspond to virus genomes, virus replication intermediates, viral transcripts, or self-RNA cleaved by RNase L. However, the relative contribution of each of these RNA species to RIG-I activation and interferon induction in virus-infected cells is not known. Here, we use three approaches to identify physiological RIG-I agonists in cells infected with influenza A virus or Sendai virus. We show that RIG-I agonists are exclusively generated by the process of virus replication and correspond to full-length virus genomes. Therefore, nongenomic viral transcripts, short replication intermediates, and cleaved self-RNA do not contribute substantially to interferon induction in cells infected with these negative strand RNA viruses. Rather, single-stranded RNA viral genomes bearing 5'-triphosphates constitute the natural RIG-I agonists that trigger cell-intrinsic innate immune responses during infection.

Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity.

RIG-I detects invading viral RNA and activates the transcription factors NF-kappaB and IRF3 through the mitochondrial protein MAVS. Here we show that RNA bearing 5'-triphosphate strongly activates the RIG-I-IRF3 signaling cascade in a reconstituted system composed of RIG-I, mitochondria, and cytosol. Activation of RIG-I requires not only RNA but also polyubiquitin chains linked through lysine 63 (K63) of ubiquitin. RIG-I binds specifically to K63-polyubiquitin chains through its tandem CARD domains in a manner that depends on RNA and ATP. Mutations in the CARD domains that abrogate ubiquitin binding also impair RIG-I activation. Remarkably, unanchored K63-ubiquitin chains, which are not conjugated to any target protein, potently activate RIG-I. These ubiquitin chains function as an endogenous ligand of RIG-I in human cells. Our results delineate the mechanism of RIG-I activation, identify CARD domains as a ubiquitin sensor, and demonstrate that unanchored K63-polyubiquitin chains are signaling molecules in antiviral innate immunity.

Antiviral Immunity and Circular RNA: No End in Sight.

In this issue of Molecular Cell, two papers by Chen et al. (2017) and Li et al. (2017) describe new insights into circRNA biogenesis and function, connecting circRNAs to innate immune pathways.

The dexd/H-box helicase DHX40 is a novel negative regulator during GCRV infection via targeting the host RLR helicases and viral nonstructural protein NS38.

RLR helicases RIG-I and MDA5, which are known as pattern recognition receptors to sense cytoplasmic viral RNAs and trigger antiviral immune responses, are DExD/H-box helicases. In teleost, whether and how non-RLR helicases regulate RLR helicases to affect viral infection remains unclear. Here, we report that the non-RLR helicase DHX40 from grass carp (namely gcDHX40) is a negative regulator of grass carp reovirus (GCRV) infection and RLR-mediated type I IFN production. GcDHX40 was a cytoplasmic protein. Ectopic expression of gcDHX40 facilitated GCRV replication and suppressed type I IFN production induced by GCRV infection and by those genes involved the RLR antiviral signaling pathway. Mechanistically, gcDHX40 promoted the generation of viral inclusion bodies (VIBs) by interacting with the NS38 protein of GCRV. Additionally, gcDHX40 interacted with RLR helicase, and impaired the formation of RLR-MAVS functional complexes. Taken together, our results indicate that gcDHX40 is a novel important proviral host factor involving in promoting the generation of GCRV VIBs and inhibiting the production of RLR-mediated type I IFNs.

The thymic epithelial microRNA network elevates the threshold for infection-associated thymic involution via miR-29a mediated suppression of the IFN-α receptor.

Thymic output is a dynamic process, with high activity at birth punctuated by transient periods of involution during infection. Interferon-α (IFN-α) is a critical molecular mediator of pathogen-induced thymic involution, yet despite the importance of thymic involution, relatively little is known about the molecular integrators that establish sensitivity. Here we found that the microRNA network dependent on the endoribonuclease Dicer, and specifically microRNA miR-29a, was critical for diminishing the sensitivity of the thymic epithelium to simulated infection signals, protecting the thymus against inappropriate involution. In the absence of Dicer or the miR-29a cluster in the thymic epithelium, expression of the IFN-α receptor by the thymic epithelium was higher, which allowed suboptimal signals to trigger rapid loss of thymic cellularity.

Ribose 2'-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5.

The 5' cap structures of higher eukaryote mRNAs have ribose 2'-O-methylation. Likewise, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases to autonomously modify their mRNAs. However, a defined biological role for 2'-O-methylation of mRNA remains elusive. Here we show that 2'-O-methylation of viral mRNA was critically involved in subverting the induction of type I interferon. We demonstrate that human and mouse coronavirus mutants lacking 2'-O-methyltransferase activity induced higher expression of type I interferon and were highly sensitive to type I interferon. Notably, the induction of type I interferon by viruses deficient in 2'-O-methyltransferase was dependent on the cytoplasmic RNA sensor Mda5. This link between Mda5-mediated sensing of viral RNA and 2'-O-methylation of mRNA suggests that RNA modifications such as 2'-O-methylation provide a molecular signature for the discrimination of self and non-self mRNA.

ZAPS is a potent stimulator of signaling mediated by the RNA helicase RIG-I during antiviral responses.

The poly(ADP-ribose) polymerases (PARPs) participate in many biological and pathological processes. Here we report that the PARP-13 shorter isoform (ZAPS), rather than the full-length protein (ZAP), was selectively induced by 5'-triphosphate-modified RNA (3pRNA) and functioned as a potent stimulator of interferon responses in human cells mediated by the RNA helicase RIG-I. ZAPS associated with RIG-I to promote the oligomerization and ATPase activity of RIG-I, which led to robust activation of IRF3 and NF-κB transcription factors. Disruption of the gene encoding ZAPS resulted in impaired induction of interferon-α (IFN-α), IFN-ß and other cytokines after viral infection. These results indicate that ZAPS is a key regulator of RIG-I signaling during the innate antiviral immune response, which suggests its possible use as a therapeutic target for viral control.

Antiviral activity of human OASL protein is mediated by enhancing signaling of the RIG-I RNA sensor.

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.

γHV68 vGAT: a viral pseudoenzyme pimping for PAMPs.

In this issue, He et al. (2015) show how herpes virus usurps a cellular metabolic enzyme to induce RIG-I deamidation and RNA-independent activation, likely to better prevent further innate immune responses.

Viral pseudo-enzymes activate RIG-I via deamidation to evade cytokine production.

RIG-I is a pattern recognition receptor that senses viral RNA and is crucial for host innate immune defense. Here, we describe a mechanism of RIG-I activation through amidotransferase-mediated deamidation. We show that viral homologs of phosphoribosylformylglycinamidine synthetase (PFAS), although lacking intrinsic enzyme activity, recruit cellular PFAS to deamidate and activate RIG-I. Accordingly, depletion and biochemical inhibition of PFAS impair RIG-I deamidation and concomitant activation. Purified PFAS and viral homolog thereof deamidate RIG-I in vitro. Ultimately, herpesvirus hijacks activated RIG-I to avoid antiviral cytokine production; loss of RIG-I or inhibition of RIG-I deamidation results in elevated cytokine production. Together, these findings demonstrate a surprising mechanism of RIG-I activation that is mediated by an enzyme.

Roquin binds inducible costimulator mRNA and effectors of mRNA decay to induce microRNA-independent post-transcriptional repression.

The molecular mechanism by which roquin controls the expression of inducible costimulator (ICOS) to prevent autoimmunity remains unsolved. Here we show that in helper T cells, roquin localized to processing (P) bodies and downregulated ICOS expression. The repression was dependent on the RNA helicase Rck, and roquin interacted with Rck and the enhancer of decapping Edc4, which act together in mRNA decapping. Sequences in roquin that confer P-body localization were essential for roquin-mediated ICOS repression. However, this process did not require microRNAs or the RNA-induced silencing complex (RISC). Instead, roquin bound ICOS mRNA directly, showing an intrinsic preference for a previously unrecognized sequence in the 3' untranslated region (3' UTR). Our results support a model in which roquin controls ICOS expression through binding to the 3' UTR of ICOS mRNA and by interacting with proteins that confer post-transcriptional repression.

RNA helicase DHX33 puts a new twist on NLRP3 inflammasome activation.

The mechanisms by which NLRP3 senses inflammasome-activating stimuli remain poorly defined. In this issue of Immunity, Mitoma et al. (2013) demonstrate that the RNA helicase DHX33 binds to cytosolic dsRNAs to trigger NLRP3 inflammasome activation.