Physiological functions of RIG-I-like receptors.

RIG-I-like receptors (RLRs) are crucial for pathogen detection and triggering immune responses and have immense physiological importance. In this review, we first summarize the interferon system and innate immunity, which constitute primary and secondary responses. Next, the molecular structure of RLRs and the mechanism of sensing non-self RNA are described. Usually, self RNA is refractory to the RLR; however, there are underlying host mechanisms that prevent immune reactions. Studies have revealed that the regulatory mechanisms of RLRs involve covalent molecular modifications, association with regulatory factors, and subcellular localization. Viruses have evolved to acquire antagonistic RLR functions to escape the host immune reactions. Finally, the pathologies caused by the malfunction of RLR signaling are described.

Caspase-mediated processing of TRBP regulates apoptosis during viral infection.

RNA silencing is a post-transcriptional gene-silencing mechanism mediated by microRNAs (miRNAs). However, the regulatory mechanism of RNA silencing during viral infection is unclear. TAR RNA-binding protein (TRBP) is an enhancer of RNA silencing that induces miRNA maturation by interacting with the ribonuclease Dicer. TRBP interacts with a virus sensor protein, laboratory of genetics and physiology 2 (LGP2), in the early stage of viral infection of human cells. Next, it induces apoptosis by inhibiting the maturation of miRNAs, thereby upregulating the expression of apoptosis regulatory genes. In this study, we show that TRBP undergoes a functional conversion in the late stage of viral infection. Viral infection resulted in the activation of caspases that proteolytically processed TRBP into two fragments. The N-terminal fragment did not interact with Dicer but interacted with type I interferon (IFN) signaling modulators, such as protein kinase R (PKR) and LGP2, and induced ER stress. The end results were irreversible apoptosis and suppression of IFN signaling. Our results demonstrate that the processing of TRBP enhances apoptosis, reducing IFN signaling during viral infection.

The Amino Acid at Position 95 in the Matrix Protein of Rabies Virus Is Involved in Antiviral Stress Granule Formation in Infected Cells.

Stress granules (SGs) are dynamic structures that store cytosolic messenger ribonucleoproteins. SGs have recently been shown to serve as a platform for activating antiviral innate immunity; however, several pathogenic viruses suppress SG formation to evade innate immunity. In this study, we investigated the relationship between rabies virus (RABV) virulence and SG formation, using viral strains with different levels of virulence. We found that the virulent Nishigahara strain did not induce SG formation, but its avirulent offshoot, the Ni-CE strain, strongly induced SG formation. Furthermore, we demonstrated that the amino acid at position 95 in the RABV matrix protein (M95), a pathogenic determinant for the Nishigahara strain, plays a key role in inhibiting SG formation, followed by protein kinase R (PKR)-dependent phosphorylation of the α subunit of eukaryotic initiation factor 2α (eIF2α). M95 was also implicated in the accumulation of RIG-I, a viral RNA sensor protein, in SGs and in the subsequent acceleration of interferon induction. Taken together, our findings strongly suggest that M95-related inhibition of SG formation contributes to the pathogenesis of RABV by allowing the virus to evade the innate immune responses of the host. IMPORTANCE Rabies virus (RABV) is a neglected zoonotic pathogen that causes lethal infections in almost all mammalian hosts, including humans. Recently, RABV has been reported to induce intracellular formation of stress granules (SGs), also known as platforms that activate innate immune responses. However, the relationship between SG formation capacity and pathogenicity of RABV has remained unclear. In this study, by comparing two RABV strains with completely different levels of virulence, we found that the amino acid mutation from valine to alanine at position 95 of matrix protein (M95), which is known to be one of the amino acid mutations that determine the difference in virulence between the strains, plays a major role in SG formation. Importantly, M95 was involved in the accumulation of RIG-I in SGs and in promoting interferon induction. These findings are the first report of the effect of a single amino acid substitution associated with SGs on viral virulence.

Epidermal clearance of Candida albicans is mediated by IL-17 but independent of fungal innate immune receptors.

IL-17 plays important roles in host defense against Candida albicans at barrier surfaces and during invasive infection. However, the role of IL-17 in host defense after colonization of the epidermis, a main site of C. albicans infection, remains poorly understood. Using a murine model of epicutaneous candidiasis without skin abrasion, we found that skin inflammation triggered by epidermal C. albicans colonization was self-limiting with fungal clearance completed by day 7 after inoculation in wild-type mice or animals deficient in IL-17A or IL-17F. In contrast, marked neutrophilic inflammation in the epidermis and impaired fungal clearance were observed in mice lacking both IL-17A and IL-17F. Clearance of C. albicans was independent of Dectin-1, Dectin-2, CARD9 (caspase-recruitment domain family, member 9), TLR2 (Toll-like receptor 2) and MyD88 in the epidermal colonization model. We found that group 3 innate lymphoid cells (ILC3s) and γδT cells were the major IL-17 producers in the epicutaneous candidiasis model. Analyses of Rag2-/- mice and Rag2-/-Il2rg-/- mice revealed that production of IL-17A and IL-17F by ILC3s was sufficient for C. albicans clearance. Finally, we found that depletion of neutrophils impaired C. albicans clearance in the epidermal colonization model. Taken together, these findings indicate a critical and redundant function of IL-17A and IL-17F produced by ILC3s in host defense against C. albicans in the epidermis. The results also suggest that epidermal C. albicans clearance is independent of innate immune receptors or that these receptors act redundantly in fungal recognition and clearance.

Dectin-2 is a direct receptor for mannose-capped lipoarabinomannan of mycobacteria.

Mycobacteria possess various immunomodulatory molecules on the cell wall. Mannose-capped lipoarabinomannan (Man-LAM), a major lipoglycan of Mycobacterium tuberculosis, has long been known to have both inhibitory and stimulatory effects on host immunity. However, the direct Man-LAM receptor that explains its pleiotropic activities has not been clearly identified. Here, we report that a C-type lectin receptor Dectin-2 (gene symbol Clec4n) is a direct receptor for Man-LAM. Man-LAM activated bone-marrow-derived dendritic cells (BMDCs) to produce pro- and anti-inflammatory cytokines, whereas it was completely abrogated in Clec4n(-/-) BMDCs. Man-LAM promoted antigen-specific T cell responses through Dectin-2 on DCs. Furthermore, Man-LAM induced experimental autoimmune encephalitis (EAE) as an adjuvant in mice, whereas Clec4n(-/-) mice were resistant. Upon mycobacterial infection, Clec4n(-/-) mice showed augmented lung pathology. These results demonstrate that Dectin-2 contributes to host immunity against mycobacterial infection through the recognition of Man-LAM.

LGP2 virus sensor enhances apoptosis by upregulating apoptosis regulatory genes through TRBP-bound miRNAs during viral infection.

During viral infection, viral nucleic acids are detected by virus sensor proteins including toll-like receptor 3 or retinoic acid-inducible gene I-like receptors (RLRs) in mammalian cells. Activation of these virus sensor proteins induces type-I interferon production and represses viral replication. Recently, we reported that an RLR family member, laboratory of genetics and physiology 2 (LGP2), modulates RNA silencing by interacting with an RNA silencing enhancer, TAR-RNA binding protein (TRBP). However, the biological implications remained unclear. Here, we show that LGP2 enhances apoptosis by upregulating apoptosis regulatory genes during viral infection. Sendai virus (SeV) infection increased LGP2 expression approximately 900 times compared to that in non-virus-infected cells. Then, the induced LGP2 interacted with TRBP, resulting in the inhibition of maturation of the TRBP-bound microRNA (miRNA) and its subsequent RNA silencing activity. Gene expression profiling revealed that apoptosis regulatory genes were upregulated during SeV infection: caspases-2, -8, -3 and -7, four cysteine proteases with key roles in apoptosis, were upregulated directly or indirectly through the repression of a typical TRBP-bound miRNA, miR-106b. Our findings may shed light on the mechanism of apoptosis, induced by the TRBP-bound miRNAs through the interaction of TRBP with LGP2, as an antiviral defense system in mammalian cells.

LGP2 virus sensor regulates gene expression network mediated by TRBP-bound microRNAs.

Here we show that laboratory of genetics and physiology 2 (LGP2) virus sensor protein regulates gene expression network of endogenous genes mediated by TAR-RNA binding protein (TRBP)-bound microRNAs (miRNAs). TRBP is an enhancer of RNA silencing, and functions to recruit precursor-miRNAs (pre-miRNAs) to Dicer that processes pre-miRNA into mature miRNA. Viral infection activates the antiviral innate immune response in mammalian cells. Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I, melanoma-differentiation-associated gene 5 (MDA5), and LGP2, function as cytoplasmic virus sensor proteins during viral infection. RIG-I and MDA5 can distinguish between different types of RNA viruses to produce antiviral cytokines, including type I interferon. However, the role of LGP2 is controversial. We found that LGP2 bound to the double-stranded RNA binding sites of TRBP, resulting in inhibition of pre-miRNA binding and recruitment by TRBP. Furthermore, although it is unclear whether TRBP binds to specific pre-miRNA, we found that TRBP bound to particular pre-miRNAs with common structural characteristics. Thus, LGP2 represses specific miRNA activities by interacting with TRBP, resulting in selective regulation of target genes. Our findings show that a novel function of LGP2 is to modulate RNA silencing, indicating the crosstalk between RNA silencing and RLR signaling in mammalian cells.

Identification of a new autoinhibitory domain of interferon-beta promoter stimulator-1 (IPS-1) for the tight regulation of oligomerization-driven signal activation.

Upon viral infection, retinoic acid-inducible gene-I (RIG-I)-like receptors detect viral foreign RNAs and transmit anti-viral signals via direct interaction with the downstream mitochondrial adaptor molecule, interferon (IFN)-ß promoter stimulator-1 (IPS-1), to inhibit viral replication. Although IPS-1 is known to form prion-like oligomers on mitochondria to activate signaling, the mechanisms that regulate oligomer formation remain unclear. Here, we identified an autoinhibitory domain (AD) at amino acids 180-349 to suppress oligomerization of IPS-1 in a resting state and regulate activation of downstream signaling. Size exclusion chromatography (SEC) analysis demonstrated that AD was required to suppress auto-oligomerization of the caspase recruitment domain (CARD) of IPS-1 via intramolecular interactions. This was supported by the observation that cleavage of a peptide bond between IPS-1 CARD and AD by Tobacco Etch virus (TEV) protease relieved autoinhibition. Conversely, deletion of this domain from IPS-1 enhanced signal activation in IFN-reporter assays, suggesting that IPS-1 AD played a critical role in the regulation of IPS-1-mediated anti-viral signal activation. These findings revealed novel molecular interactions involved in the tight regulation of innate anti-viral immunity.

Fecal microbiota transplantation prevents Candida albicans from colonizing the gastrointestinal tract.

Gut microbes symbiotically colonize the gastrointestinal (GI) tract, interacting with each other and their host to maintain GI tract homeostasis. Recent reports have shown that gut microbes help protect the gut from colonization by pathogenic microbes. Here, we report that commensal microbes prevent colonization of the GI tract by the pathogenic fungus, Candida albicans. Wild-type specific pathogen-free (SPF) mice are resistant to C. albicans colonization of the GI tract. However, administering certain antibiotics to SPF mice enables C. albicans colonization. Quantitative kinetics of commensal bacteria are inversely correlated with the number of C. albicans in the gut. Here, we provide further evidence that transplantation of fecal microbiota is effective in preventing Candida colonization of the GI tract. These data demonstrate the importance of commensal bacteria as a barrier for the GI tract surface and highlight the potential clinical applications of commensal bacteria in preventing pathogenic fungal infections.

Inhibition of the Inflammatory Pathway Enhances Both the in Vitro and in Vivo Transfection Activity of Exogenous in Vitro-Transcribed mRNAs Delivered by Lipid Nanoparticles.

While the use of in vitro-transcribed mRNA (IVT-mRNA) in therapeutics is a rapidly expanding area, the transfection of the exogenous IVT-mRNA is accompanied by a risk of immune activation. This immunological defense mechanism suppresses cellular translation process and can reduce transfection efficiency to a considerable extent. In the present study, we investigated the in vitro effects of Integrated Stress Response Inhibitor (ISRIB), and dexamethasone, a steroidal anti-inflammatory drug, on the transfection activity of a lipid nanoparticle (LNP) that was composed of ionizable lipids and IVT-mRNA. In the case of transfection to mouse embryonic fibroblast (MEF) cells, ISRIB mainly enhanced the transfection activity at an early stage of transfection (0-6 h). In contrast, dexamethasone caused an increase in transfection activity at intermediate-late stages of transfection (4-48 h). We also investigated the in vivo effects of dexamethasone using an LNP on that the IVT-mRNA and lipid-conjugated dexamethasone (Dex-Pal) were co-loaded. The intravenous administration of the LNP successfully enhanced the protein expression in a mouse liver by up to 6.6-fold. Collectively, the co-delivery of an anti-inflammatory drug is a promising approach for enhancing transfection efficiency of IVT-mRNA.

Leader-Containing Uncapped Viral Transcript Activates RIG-I in Antiviral Stress Granules.

RIG-I triggers antiviral responses by recognizing viral RNA (vRNA) in the cytoplasm. However, the spatio-temporal dynamics of vRNA sensing and signal transduction remain elusive. We investigated the time course of events in cells infected with Newcastle disease virus (NDV), a non-segmented negative-strand RNA virus. RIG-I was recruited to viral replication complexes (vRC) and triggered minimal primary type I interferon (IFN) production. RIG-I subsequently localized to antiviral stress granules (avSG) induced after vRC formation. The inhibition of avSG attenuated secondary IFN production, suggesting avSG as a platform for efficient vRNA detection. avSG selectively captured positive-strand vRNA, and poly(A)+ RNA induced IFN production. Further investigations suggested that uncapped vRNA derived from read-through transcription was sensed by RIG-I in avSG. These results highlight how viral infections stimulate host stress responses, thereby selectively recruiting uncapped vRNA to avSG, in which RIG-I and other components cooperate in an efficient antiviral program.

Antiviral innate immunity and stress granule responses.

Viral infection triggers the activation of antiviral innate immune responses in mammalian cells. Viral RNA in the cytoplasm activates signaling pathways that result in the production of interferons (IFNs) and IFN-stimulated genes. Some viral infections have been shown to induce cytoplasmic granular aggregates similar to the dynamic ribonucleoprotein aggregates termed stress granules (SGs), suggesting that these viruses may utilize this stress response for their own benefit. By contrast, some viruses actively inhibit SG formation, suggesting an antiviral function for these structures. We review here the relationship between different viral infections and SG formation. We examine the evidence for antiviral functions for SGs and highlight important areas of inquiry towards understanding cellular stress responses to viral infection.

Structural mechanism of RNA recognition by the RIG-I-like receptors.

Cytoplasmic nonself RNA, such as that generated by invading viruses, is recognized by a family of sensory molecules termed RIG-I-like Receptors (RLRs). Here, we discuss the mechanism of the RLRs' sensing of nonself RNA. Our findings define three functional domains of RLRs and provide insights into how RLRs function as a molecular switch through interactions with virus-specific RNA ligands.

A novel function of human Pumilio proteins in cytoplasmic sensing of viral infection.

RIG-I-like receptor (RLR) plays a pivotal role in the detection of invading pathogens to initiate type I interferon (IFN) gene transcription. Since aberrant IFN production is harmful, RLR signaling is strictly regulated. However, the regulatory mechanisms are not fully understood. By expression cloning, we identified Pumilio proteins, PUM1 and PUM2, as candidate positive regulators of RIG-I signaling. Overexpression of Pumilio proteins and their knockdown augmented and diminished IFN-ß promoter activity induced by Newcastle disease virus (NDV), respectively. Both proteins showed a specific association with LGP2, but not with RIG-I or MDA5. Furthermore, all of these components were recruited to NDV-induced antiviral stress granules. Interestingly, biochemical analyses revealed that Pumilio increased double-stranded (ds) RNA binding affinity of LGP2; however, Pumilio was absent in the dsRNA-LGP2 complex, suggesting that Pumilio facilitates viral RNA recognition by LGP2 through its chaperon-like function. Collectively, our results demonstrate an unknown function of Pumilio in viral recognition by LGP2.

DHX36 enhances RIG-I signaling by facilitating PKR-mediated antiviral stress granule formation.

RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR).

Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses.

A DExD/H protein, RIG-I, is critical in innate antiviral responses by sensing viral RNA. Here we show that RIG-I recognizes two distinct viral RNA patterns: double-stranded (ds) and 5'ppp single-stranded (ss) RNA. The binding of RIG-I with dsRNA or 5'ppp ssRNA in the presence of ATP produces a common structure, as suggested by protease digestion. Further analyses demonstrated that the C-terminal domain of RIG-I (CTD) recognizes these RNA patterns and CTD coincides with the autorepression domain. Structural analysis of CTD by NMR spectroscopy in conjunction with mutagenesis revealed that the basic surface of CTD with a characteristic cleft interacts with RIG-I ligands. Our results suggest that the bipartite structure of CTD regulates RIG-I on encountering viral RNA patterns.

Encephalomyocarditis virus disrupts stress granules, the critical platform for triggering antiviral innate immune responses.

In response to stress, cells induce ribonucleoprotein aggregates, termed stress granules (SGs). SGs are transient loci containing translation-stalled mRNA, which is eventually degraded or recycled for translation. Infection of some viruses, including influenza A virus with a deletion of nonstructural protein 1 (IAVΔNS1), induces SG-like protein aggregates. Previously, we showed that IAVΔNS1-induced SGs are required for efficient induction of type I interferon (IFN). Here, we investigated SG formation by different viruses using green fluorescent protein (GFP)-tagged Ras-Gap SH3 domain binding protein 1 (GFP-G3BP1) as an SG probe. HeLa cells stably expressing GFP-G3BP1 were infected with different viruses, and GFP fluorescence was monitored live with time-lapse microscopy. SG formations by different viruses was classified into 4 different patterns: no SG formation, stable SG formation, transient SG formation, and alternate SG formation. We focused on encephalomyocarditis virus (EMCV) infection, which exhibited transient SG formation. We found that EMCV disrupts SGs by cleavage of G3BP1 at late stages of infection (>8 h) through a mechanism similar to that used by poliovirus. Expression of a G3BP1 mutant that is resistant to the cleavage conferred persistent formation of SGs as well as an enhanced induction of IFN and other cytokines at late stages of infection. Additionally, knockdown of endogenous G3BP1 blocked SG formation with an attenuated induction of IFN and potentiated viral replication. Taken together, our findings suggest a critical role of SGs as an antiviral platform and shed light on one of the mechanisms by which a virus interferes with host stress and subsequent antiviral responses.

Hepatitis C virus NS4B protein targets STING and abrogates RIG-I-mediated type I interferon-dependent innate immunity.

UNLABELLED: Hepatitis C virus (HCV) infection blocks cellular interferon (IFN)-mediated antiviral signaling through cleavage of Cardif by HCV-NS3/4A serine protease. Like NS3/4A, NS4B protein strongly blocks IFN-ß production signaling mediated by retinoic acid-inducible gene I (RIG-I); however, the underlying molecular mechanisms are not well understood. Recently, the stimulator of interferon genes (STING) was identified as an activator of RIG-I signaling. STING possesses a structural homology domain with flaviviral NS4B, which suggests a direct protein-protein interaction. In the present study, we investigated the molecular mechanisms by which NS4B targets RIG-I-induced and STING-mediated IFN-ß production signaling. IFN-ß promoter reporter assay showed that IFN-ß promoter activation induced by RIG-I or Cardif was significantly suppressed by both NS4B and NS3/4A, whereas STING-induced IFN-ß activation was suppressed by NS4B but not by NS3/4A, suggesting that NS4B had a distinct point of interaction. Immunostaining showed that STING colocalized with NS4B in the endoplasmic reticulum. Immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays demonstrated that NS4B specifically bound STING. Intriguingly, NS4B expression blocked the protein interaction between STING and Cardif, which is required for robust IFN-ß activation. NS4B truncation assays showed that its N terminus, containing the STING homology domain, was necessary for the suppression of IFN-ß promoter activation. NS4B suppressed residual IFN-ß activation by an NS3/4A-cleaved Cardif (Cardif1-508), suggesting that NS3/4A and NS4B may cooperate in the blockade of IFN-ß production. CONCLUSION: NS4B suppresses RIG-I-mediated IFN-ß production signaling through a direct protein interaction with STING. Disruption of that interaction may restore cellular antiviral responses and may constitute a novel therapeutic strategy for the eradication of HCV.

LGP2 is a positive regulator of RIG-I- and MDA5-mediated antiviral responses.

RNA virus infection is recognized by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), RIG-I, and melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm. RLRs are comprised of N-terminal caspase-recruitment domains (CARDs) and a DExD/H-box helicase domain. The third member of the RLR family, LGP2, lacks any CARDs and was originally identified as a negative regulator of RLR signaling. In the present study, we generated mice lacking LGP2 and found that LGP2 was required for RIG-I- and MDA5-mediated antiviral responses. In particular, LGP2 was essential for type I IFN production in response to picornaviridae infection. Overexpression of the CARDs from RIG-I and MDA5 in Lgp2(-/-) fibroblasts activated the IFN-beta promoter, suggesting that LGP2 acts upstream of RIG-I and MDA5. We further examined the role of the LGP2 helicase domain by generating mice harboring a point mutation of Lys-30 to Ala (Lgp2 (K30A/K30A)) that abrogated the LGP2 ATPase activity. Lgp2 (K30A/K30A) dendritic cells showed impaired IFN-beta productions in response to various RNA viruses to extents similar to those of Lgp2(-/-) cells. Lgp2(-/-) and Lgp2 (K30A/K30A) mice were highly susceptible to encephalomyocarditis virus infection. Nevertheless, LGP2 and its ATPase activity were dispensable for the responses to synthetic RNA ligands for MDA5 and RIG-I. Taken together, the present data suggest that LGP2 facilitates viral RNA recognition by RIG-I and MDA5 through its ATPase domain.