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.

Protein kinase R and the integrated stress response drive immunopathology caused by mutations in the RNA deaminase ADAR1.

The RNA deaminase ADAR1 is an essential negative regulator of the RNA sensor MDA5, and loss of ADAR1 function triggers inappropriate activation of MDA5 by self-RNAs. Mutations in ADAR, the gene that encodes ADAR1, cause human immune diseases, including Aicardi-Goutières syndrome (AGS). However, the mechanisms of MDA5-dependent disease pathogenesis in vivo remain unknown. Here we generated mice with a single amino acid change in ADAR1 that models the most common human ADAR AGS mutation. These Adar mutant mice developed lethal disease that required MDA5, the RIG-I-like receptor LGP2, type I interferons, and the eIF2α kinase PKR. A small-molecule inhibitor of the integrated stress response (ISR) that acts downstream of eIF2α phosphorylation prevented immunopathology and rescued the mice from mortality. These findings place PKR and the ISR as central components of immunopathology in vivo and identify therapeutic targets for treatment of human diseases associated with the ADAR1-MDA5 axis.

Structural analysis of RIG-I-like receptors reveals ancient rules of engagement between diverse RNA helicases and TRIM ubiquitin ligases.

RNA helicases and E3 ubiquitin ligases mediate many critical functions in cells, but their actions have largely been studied in distinct biological contexts. Here, we uncover evolutionarily conserved rules of engagement between RNA helicases and tripartite motif (TRIM) E3 ligases that lead to their functional coordination in vertebrate innate immunity. Using cryoelectron microscopy and biochemistry, we show that RIG-I-like receptors (RLRs), viral RNA receptors with helicase domains, interact with their cognate TRIM/TRIM-like E3 ligases through similar epitopes in the helicase domains. Their interactions are avidity driven, restricting the actions of TRIM/TRIM-like proteins and consequent immune activation to RLR multimers. Mass spectrometry and phylogeny-guided biochemical analyses further reveal that similar rules of engagement may apply to diverse RNA helicases and TRIM/TRIM-like proteins. Our analyses suggest not only conserved substrates for TRIM proteins but also, unexpectedly, deep evolutionary connections between TRIM proteins and RNA helicases, linking ubiquitin and RNA biology throughout animal evolution.

K63-linked polyubiquitination of LGP2 by Riplet regulates RIG-I-dependent innate immune response.

Type I interferons (IFNs) exhibit strong antiviral activity and induce the expression of antiviral proteins. Since excessive expression of type I IFNs is harmful to the host, their expression should be turned off at the appropriate time. In this study, we find that post-translational modification of LGP2, a member of the RIG-I-like receptor family, modulates antiviral innate immune responses. The LGP2 protein undergoes K63-linked polyubiquitination in response to cytoplasmic double-stranded RNAs or viral infection. Our mass spectrometry analysis reveals the K residues ubiquitinated by the Riplet ubiquitin ligase. LGP2 ubiquitination occurs with a delay compared to RIG-I ubiquitination. Interestingly, ubiquitination-defective LGP2 mutations increase the expression of type I IFN at a late phase, whereas the mutant proteins attenuate other antiviral proteins, such as SP100, PML, and ANKRD1. Our data indicate that delayed polyubiquitination of LGP2 fine-tunes RIG-I-dependent antiviral innate immune responses at a late phase of viral infection.

LGP2 Promotes Type I Interferon Production To Inhibit PRRSV Infection via Enhancing MDA5-Mediated Signaling.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the global pig industry, which modulates the host's innate antiviral immunity to achieve immune evasion. RIG-I-like receptors (RLRs) sense viral RNA and activate the interferon signaling pathway. LGP2, a member of the RLR family, plays an important role in regulating innate immunity. However, the role of LGP2 in virus infection is controversial. Whether LGP2 has a role during infection with PRRSV remains unclear. Here, we found that LGP2 overexpression restrained the replication of PRRSV, while LGP2 silencing facilitated PRRSV replication. LGP2 was prone to interact with MDA5 and enhanced viral RNA enrichment and recognition by MDA5, thus promoting the activation of RIG-I/IRF3 and NF-κB signaling pathways and reinforcing the expression of proinflammatory cytokines and type I interferon during PRRSV infection. Meanwhile, there was a decreased protein expression of LGP2 upon PRRSV infection in vitro. PRRSV Nsp1 and Nsp2 interacted with LGP2 and promoted K63-linked ubiquitination of LGP2, ultimately leading to the degradation of LGP2. These novel findings indicate that LGP2 plays a role in regulating PRRSV replication through synergistic interaction with MDA5. Moreover, targeting LGP2 is responsible for PRRSV immune evasion. Our work describes a novel mechanism of virus-host interaction and provides the basis for preventing and controlling PRRSV. IMPORTANCE LGP2, a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), shows higher-affinity binding to RNA and work synergism with RIG-I or MDA5. However, LGP2 has divergent responses to different viruses, which remains controversial in antiviral immune responses. Here, we present the detailed process of LGP2 in positively regulating the anti-PRRSV response. Upon PRRSV infection, LGP2 was prone to bind to MDA5 and enhanced MDA5 signaling, manifesting the enrichment of viral RNA on MDA5 and the activation of downstream IRF3 and NF-κB, which results in increased proinflammatory cytokines and type I interferon expression, ultimately inhibiting PRRSV at the early stage of infection. Moreover, PRRSV Nsp1 and Nsp2 interacted with LGP2 via ubiquitin-proteasome pathways, thus blocking LGP2-mediated immune response. This research helps us understand the host recognition and innate antiviral response to PRRSV infection by neglected pattern recognition receptors, which sheds light on the detailed mechanism of virus-host interaction.

ANAX4 is a downstream molecule of LGP2 and promotes GCRV proliferation.

This study explored the key molecules and signal pathways in the pathogenesis of grass carp reovirus (GCRV). Using immunoprecipitation mass spectrometry and Co-IP validation, the protein CiANXA4 was identified which interacts indirectly with CiLGP2. CiANXA4 encodes 321 amino acids, including 4 ANX domains. To explore the role of CiANXA4 in the anti-GCRV immune response, we used overexpression and siRNA knockdown in cells. The results showed that overexpression of the CiANXA4 gene significantly increased the mRNA content of vp2 and vp7 in GCRV-infected cells, and the virus titer greatly increased. Knockdown of CiANXA4 significantly inhibited the mRNA levels of vp2 and vp7, and the protein levels of viral protein VP7 also significantly decreased. This suggests that CiANXA4 promotes viral proliferation. Further, we demonstrate that the ANX3 and ANX4 domains are key domains that limit CiANXA4 function by constructing domain-deletion mutants. Finally, we investigated the relationship between CiLGP2 and CiANXA4. RT-PCR and Western blot results showed that CiLGP2 mRNA and protein expression levels were not affected by CiANXA4 overexpression. In contrast, overexpression of CiLGP2 resulted in significant reductions in CiANXA4 mRNA and protein levels. This suggests that the function of CiANXA4 is restricted by CiLGP2, and CiANXA4 is a downstream molecule of CiLGP2. These results reveal that CiANXA4 plays a critical role in the anti-GCRV innate immune response of grass carp, and provides new targets and strategies to develop antiviral drugs and improve disease resistance in grass carp.

Comparison of HAV and HCV infections in vivo and in vitro reveals distinct patterns of innate immune evasion and activation.

BACKGROUND & AIMS: Hepatitis A virus (HAV) infections are considered not to trigger innate immunity in vivo, in contrast to hepatitis C virus (HCV). This lack of induction has been imputed to strong interference by HAV proteases 3CD and 3ABC. We aimed to elucidate the mechanisms of immune activation and counteraction by HAV and HCV in vivo and in vitro. METHODS: Albumin-urokinase-type plasminogen activator/severe combined immunodeficiency (Alb/uPA-SCID) mice with humanised livers were infected with HAV and HCV. Hepatic cell culture models were used to assess HAV and HCV sensing by Toll-like receptor 3 and retinoic acid-inducible gene I/melanoma differentiation-associated protein 5 (RIG-I/MDA5), respectively. Cleavage of the adaptor proteins TIR-domain-containing adapter-inducing interferon-ß (TRIF) and mitochondrial antiviral-signalling protein (MAVS) was analysed by transient and stable expression of HAV and HCV proteases and virus infection. RESULTS: We detected similar levels of interferon-stimulated gene induction in hepatocytes of HAV- and HCV-infected mice with humanised liver. In cell culture, HAV induced interferon-stimulated genes exclusively upon MDA5 sensing and depended on LGP2 (laboratory of genetics and physiology 2). TRIF and MAVS were only partially cleaved by HAV 3ABC and 3CD, not sufficiently to abrogate signalling. In contrast, HCV NS3-4A efficiently degraded MAVS, as previously reported, whereas TRIF cleavage was not detected. CONCLUSIONS: HAV induces an innate immune response in hepatocytes via MDA5/LGP2, with limited control of both pathways by proteolytic cleavage. HCV activates Toll-like receptor 3 and lacks TRIF cleavage, suggesting that this pathway mainly contributes to HCV-induced antiviral responses in hepatocytes. Our results shed new light on the induction of innate immunity and counteraction by HAV and HCV. IMPACT AND IMPLICATIONS: Understanding the mechanisms that determine the differential outcomes of HAV and HCV infections is crucial for the development of effective therapies. Our study provides insights into the interplay between these viruses and the host innate immune response in vitro and in vivo, shedding light on previously controversial or only partially investigated aspects. This knowledge could tailor the development of new strategies to combat HCV persistence, as well as improve our understanding of the factors underlying successful HAV clearance.

Black carp LGP2 suppresses RIG-I mediated IFN signaling during the antiviral innate immunity.

Laboratory of genetics and physiology 2 (LGP2), a member of retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), has been reported to play different roles in IFN signaling in both mammals and teleost fish. In our previous study, black carp (Mylopharyngodon piceus) LGP2 (bcLGP2) has been characterized to positively regulate melanoma differentiation-associated gene 5 (MDA5). In this study, knockdown of bcLGP2 decreased the expression of host genes, including bcIFNb, bcPKR, bcMx1, and bcViperin, and also attenuated the antiviral capability of host cells. The relationship between bcLGP2 and black carp RIG-Ib (bcRIG-Ib) has been explored. Dual-luciferase reporter assay and qRT-PCR assay indicated that bcLGP2 dampened bcRIG-Ib induced transcription of type I interferons (IFNs) and interferon-stimulated genes (ISGs), including PKR, ISG15, and Viperin. Consistently, the plaque assay identified that bcLGP2 attenuated bcRIG-Ib mediated antiviral ability against spring viremia of carp virus (SVCV). Co-immunoprecipitation assay identified the interaction between bcLGP2 and bcRIG-Ib, as well as bcLGP2 and bcRIG-Ib-CARD. And bcRIG-Ib-CARD mediated antiviral ability was also attenuated by bcLGP2. Truncation mutation analysis showed DExD/H-box Helicase domain of bcLGP2 possessed a similar inhibitory effect on bcRIG-Ib to that of bcLGP2, while the C-terminus repressor domain (CTD) presented little impact on bcRIG-Ib. Furthermore, bcLGP2 enhanced the K48-linked ubiquitination of bcRIG-Ib, promoting proteasome-dependent degradation of bcRIG-Ib. Thus, our data supported the conclusion that bcLGP2 interacted with and induced degradation of bcRIG-Ib through proteasome, leading to the dampened antiviral signaling mediated by bcRIG-Ib.

Spatiotemporal dynamics of innate immune signaling via RIG-I-like receptors.

RIG-I, MDA5, and LGP2 comprise the RIG-I-like receptors (RLRs). RIG-I and MDA5 are essential pathogen recognition receptors sensing viral infections while LGP2 has been described as both RLR cofactor and negative regulator. After sensing and binding to viral RNA, including double-stranded RNA (dsRNA), RIG-I and MDA5 undergo cytosol-to-membrane relocalization to bind and signal through the MAVS adaptor protein on intracellular membranes, thus directing downstream activation of IRF3 and innate immunity. Here, we report examination of the dynamic subcellular localization of all three RLRs within the intracellular response to dsRNA and RNA virus infection. Observations from high resolution biochemical fractionation and electron microscopy, coupled with analysis of protein interactions and IRF3 activation, show that, in resting cells, microsome but not mitochondrial fractions harbor the central components to initiate innate immune signaling. LGP2 interacts with MAVS in microsomes, blocking the RIG-I/MAVS interaction. Remarkably, in response to dsRNA treatment or RNA virus infection, LGP2 is rapidly released from MAVS and redistributed to mitochondria, temporally correlating with IRF3 activation. We reveal that IRF3 activation does not take place on mitochondria but instead occurs at endoplasmic reticulum (ER)-derived membranes. Our observations suggest ER-derived membranes as key RLR signaling platforms controlled through inhibitory actions of LGP2 binding to MAVS wherein LGP2 translocation to mitochondria releases MAVS inhibition to facilitate RLR-mediated signaling of innate immunity.

The first identified invertebrate LGP2-like homolog gene in the Pacific oyster Crassostrea gigas.

The LGP2 (Laboratory of Genetics and Physiology 2) protein is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLRs) family, which is a class of antiviral pattern recognition receptors located in the cytoplasm. However, few studies have investigated the function of LGP2 in invertebrates. In this study, the complete coding sequence of the LGP2 gene of the Pacific oyster, Crassostrea gigas, was obtained and named CgLGP2-like. Sequence analysis revealed that CgLGP2-like encodes 803 amino acids, and the encoded protein contains a DEXDc, HELICc, and C-terminal regulatory domains. Multiple sequence alignment demonstrated that the sequences of these key protein functional domains were relatively conserved. Phylogenetic analysis revealed that CgLGP2-like was a new member of the animal LGP2 family. Quantitative real-time PCR results showed that CgLGP2-like mRNA was expressed in all tested oyster tissues, with the highest expression observed in the labial palpus and digestive glands. CgLGP2-like expression in gill tissues was significantly induced after the poly(I:C) challenge. Furthermore, multiple IRF and NF-κB binding sites were identified in the CgLGP2-like promoter region, which may be one of the reasons why CgLGP2-like responds to poly(I:C) stimulation. Finally, the results of dual-luciferase reporter gene assays revealed that overexpression of CgLGP2-like may have a regulatory effect on the human IFN, AP-1, and oyster CgIL-17 genes in HEK293T cells. Overall, our results preliminarily elucidate the immune functions of invertebrate LGP2 protein and provide valuable information for the development of comparative immunology.

DDX39B interacts with the pattern recognition receptor pathway to inhibit NF-κB and sensitize to alkylating chemotherapy.

BACKGROUND: Nuclear factor-κB (NF-κB) plays a prominent role in promoting inflammation and resistance to DNA damaging therapy. We searched for proteins that modulate the NF-κB response as a prerequisite to identifying novel factors that affect sensitivity to DNA damaging chemotherapy. RESULTS: Using streptavidin-agarose pull-down, we identified the DExD/H-box RNA helicase, DDX39B, as a factor that differentially interacts with κB DNA probes. Subsequently, using both RNA interference and CRISPR/Cas9 technology, we demonstrated that DDX39B inhibits NF-κB activity by a general mechanism involving inhibition of p65 phosphorylation. Mechanistically, DDX39B mediates this effect by interacting with the pattern recognition receptor (PRR), LGP2, a pathway that required the cellular response to cytoplasmic double-stranded RNA (dsRNA). From a functional standpoint, loss of DDX39B promoted resistance to alkylating chemotherapy in glioblastoma cells. Further examination of DDX39B demonstrated that its protein abundance was regulated by site-specific sumoylation that promoted its poly-ubiquitination and degradation. These post-translational modifications required the presence of the SUMO E3 ligase, PIASx-ß. Finally, genome-wide analysis demonstrated that despite the link to the PRR system, DDX39B did not generally inhibit interferon-stimulated gene expression, but rather acted to attenuate expression of factors associated with the extracellular matrix, cellular migration, and angiogenesis. CONCLUSIONS: These results identify DDX39B, a factor with known functions in mRNA splicing and nuclear export, as an RNA-binding protein that blocks a subset of the inflammatory response. While these findings identify a pathway by which DDX39B promotes sensitization to DNA damaging therapy, the data also reveal a mechanism by which this helicase may act to mitigate autoimmune disease.

RNA sensor LGP2 inhibits TRAF ubiquitin ligase to negatively regulate innate immune signaling.

The production of type I interferon (IFN) is essential for cellular barrier functions and innate and adaptive antiviral immunity. In response to virus infections, RNA receptors RIG-I and MDA5 stimulate a mitochondria-localized signaling apparatus that uses TRAF family ubiquitin ligase proteins to activate master transcription regulators IRF3 and NFκB, driving IFN and antiviral target gene expression. Data indicate that a third RNA receptor, LGP2, acts as a negative regulator of antiviral signaling by interfering with TRAF family proteins. Disruption of LGP2 expression in cells results in earlier and overactive transcriptional responses to virus or dsRNA LGP2 associates with the C-terminus of TRAF2, TRAF3, TRAF5, and TRAF6 and interferes with TRAF ubiquitin ligase activity. TRAF interference is independent of LGP2 ATP hydrolysis, RNA binding, or its C-terminal domain, and LGP2 can regulate TRAF-mediated signaling pathways in trans, including IL-1ß, TNFα, and cGAMP These findings provide a unique mechanism for LGP2 negative regulation through TRAF suppression and extend the potential impact of LGP2 negative regulation beyond the IFN antiviral response.

PUM1 is a biphasic negative regulator of innate immunity genes by suppressing LGP2.

PUM1 is an RNA binding protein shown to regulate the stability and function of mRNAs bearing a specific sequence. We report the following: (i) A key function of PUM1 is that of a repressor of key innate immunity genes by repressing the expression of LGP2. Thus, between 12 and 48 hours after transfection of human cells with siPUM1 RNA there was an initial (phase 1) upsurge of transcripts encoding LGP2, CXCL10, IL6, and PKR. This was followed 24 hours later (phase 2) by a significant accumulation of mRNAs encoding RIG-I, SP100, MDA5, IFIT1, PML, STING, and IFNß. The genes that were not activated encoded HDAC4 and NF-κB1. (ii) Simultaneous depletion of PUM1 and LGP2, CXCL10, or IL6 revealed that up-regulation of phase 1 and phase 2 genes was the consequence of up-regulation of LGP2. (iii) IFNß produced 48-72 hours after transfection of siPUM1 was effective in up-regulating LGP2 and phase 2 genes and reducing the replication of HSV-1 in untreated cells. (iv) Because only half of genes up-regulated in phase 1 and 2 encode mRNAs containing PUM1 binding sites, the upsurge in gene expression could not be attributed solely to stabilization of mRNAs in the absence of PUM1. (v) Lastly, depletion of PUM2 does not result in up-regulation of phase 1 or phase 2 genes. The results of the studies presented here indicate that PUM1 is a negative regulator of LGP2, a master regulator of innate immunity genes expressed in a cascade fashion.

Molecular characterization and expressional quantification of lgp2, a modulatory co-receptor of RLR-signalling pathway in the Indian major carp Labeo rohita following pathogenic challenges and PAMP stimulations.

Lgp2 (laboratory of genetics and physiology 2) is a cytosolic viral sensor of the RLR (retinoic acid-inducible gene 1 like receptor) family member without the caspase recruitment domain, having both inhibitory and stimulatory roles in RLR-signalling pathway. In India, Labeo rohita (rohu) is one of the leading and economically favoured freshwater fish species. Several immunological sentry proteins have been reported in this fish species, but no information is available on the RLR members. This study was aimed at cloning and characterization of full-length lgp2-cDNA (complementary DNA) in rohu and investigation of its expressional modulations following various pathogen-associated molecular pattern stimulations and bacterial infections. The full-length lgp2-cDNA sequence obtained through rapid amplification of cDNA ends-PCR consisted of 2299 nucleotides with an open reading frame of 2034 bp encoding 677 amino acids. In rohu-Lgp2, four conserved domains - a DEAD/DEAH box helicase domain, Pfam type-III restriction enzyme domain, helicase superfamily c-terminal domain and RIG-I C-terminal regulatory domain - have been detected. Within these domains, several important functional motifs, such as ATP-binding site, ATPase motif, RNA unwinding motif and RNA-binding sites, have also been identified. In healthy rohu, lgp2 gene was abundantly expressed in gill, liver, kidney, spleen and blood. In response to both in vitro and in vivo treatments using double-stranded RNA (poly I:C), lgp2 gene expression was significantly (P < 0.05) upregulated in all tested tissues and also in the LRG (Labeo rohita gill) cells. lgp2 gene expression significantly (P < 0.05) increased on stimulation of LRG cells using γ-d-glutamyl-meso-diaminopimelic acid and muramyl dipeptide. In vivo treatment using lipopolysaccharide and Aeromonas hydrophila-derived RNA resulted in both up- and down-regulation of lgp2 gene expression. Upon gram-positive and gram-negative bacterial infections, the expression of the lgp2 gene increased at different times in almost all the tested tissues. These integrated observations in rohu suggest that Lgp2 is an antiviral and antibacterial cytosolic receptor. SIGNIFICANCE STATEMENT: Lgp2, a cytosolic viral sensor of retinoic acid-inducible gene 1 like receptor family member, has been cloned in Labeo rohita. The complete sequence of rohu lgp2-complementary DNA consisted of 2299 nucleotides with an open reading frame of 2034 bp encoding 677 amino acids. It consisted of a DExDc, RES-III, HELICc, Pfam RIG-I_C-RD, ATP-binding site, ATPase motif, RNA unwinding motif and RNA-binding site. Upon bacterial infection, double-stranded RNA and various pathogen-associated molecular pattern stimulations, lgp2 gene expression significantly increased, indicating its role as an antiviral and antibacterial cytosolic receptor.

Structures of RIG-I-Like Receptors and Insights into Viral RNA Sensing.

RIG-I-like receptors (RLRs) are an important family of pattern recognition receptors. They activate the immunological responses against viral infections by sensing RNAs in the cytoplasm. Recent studies provide significant insights into the activation and transduction mechanisms of RLRs family (members including RIG-I, MDA5, and LGP2). Here we review our current understanding of the structures of RLRs. Structural characterizations of RLRs family have revealed the mechanism of their actions at molecular level. The activation mechanisms of RIG-I and MDA5 are different, despite both of them have similar domain organizations and bind to dsRNA ligands. RIG-I, but not MDA5, adopts an auto-suppression conformation in the absence of RNA. In addition to ligand triggered receptor oligomerization, the activities of these receptors are also regulated by several posttranslational modifications, especially ubiquitination. Overall, these structural studies play critical roles in promoting the understanding of viral RNA recognition mechanisms by the host innate immune system, which also contribute to the designing of drugs for treatment of viral infection. However, much work remains to be done in studying the signaling pathway of MDA5 and LGP2, particularly by structural biology.

New Techniques to Study Intracellular Receptors in Living Cells: Insights Into RIG-I-Like Receptor Signaling.

This review discusses new developments in Förster resonance energy transfer (FRET) microscopy and its application to cellular receptors. The method is based on the kinetic theory of FRET, which can be used to predict FRET not only in dimers, but also higher order oligomers of donor and acceptor fluorophores. Models based on such FRET predictions can be fit to observed FRET efficiency histograms (also called FRET spectrograms) and used to estimate intracellular binding constants, free energy values, and stoichiometries. These "FRET spectrometry" methods have been used to analyze oligomers formed by various receptors in cell signaling pathways, but until recently such studies were limited to receptors residing on the cell surface. To study complexes residing inside the cell, a technique called Quantitative Micro-Spectroscopic Imaging (Q-MSI) was developed. Q-MSI combines determination of quaternary structure from pixel-level apparent FRET spectrograms with the determination of both donor and acceptor concentrations at the organelle level. This is done by resolving and analyzing the spectrum of a third fluorescent marker, which does not participate in FRET. Q-MSI was first used to study the interaction of a class of cytoplasmic receptors that bind viral RNA and signal an antiviral response via complexes formed mainly on mitochondrial membranes. Q-MSI revealed previously unknown RNA mitochondrial receptor orientations, and the interaction between the viral RNA receptor called LGP2 with the RNA helicase encoded by the hepatitis virus. The biological importance of these new observations is discussed.

Quantitative microspectroscopic imaging reveals viral and cellular RNA helicase interactions in live cells.

Human cells detect RNA viruses through a set of helicases called RIG-I-like receptors (RLRs) that initiate the interferon response via a mitochondrial signaling complex. Many RNA viruses also encode helicases, which are sometimes covalently linked to proteases that cleave signaling proteins. One unresolved question is how RLRs interact with each other and with viral proteins in cells. This study examined the interactions among the hepatitis C virus (HCV) helicase and RLR helicases in live cells with quantitative microspectroscopic imaging (Q-MSI), a technique that determines FRET efficiency and subcellular donor and acceptor concentrations. HEK293T cells were transfected with various vector combinations to express cyan fluorescent protein (CFP) or YFP fused to either biologically active HCV helicase or one RLR (i.e. RIG-I, MDA5, or LGP2), expressed in the presence or absence of polyinosinic-polycytidylic acid (poly(I:C)), which elicits RLR accumulation at mitochondria. Q-MSI confirmed previously reported RLR interactions and revealed an interaction between HCV helicase and LGP2. Mitochondria in CFP-RIG-I:YFP-RIG-I cells, CFP-MDA5:YFP-MDA5 cells, and CFP-MDA5:YFP-LGP2 cells had higher FRET efficiencies in the presence of poly(I:C), indicating that RNA causes these proteins to accumulate at mitochondria in higher-order complexes than those formed in the absence of poly(I:C). However, mitochondria in CFP-LGP2:YFP-LGP2 cells had lower FRET signal in the presence of poly(I:C), suggesting that LGP2 oligomers disperse so that LGP2 can bind MDA5. Data support a new model where an LGP2-MDA5 oligomer shuttles NS3 to the mitochondria to block antiviral signaling.

Innate immune sensor laboratory of genetics and physiology 2 suppresses tumor cell growth and functions as a prognostic marker in neuroblastoma.

The innate immune receptors, such as toll-like receptor 3 (TLR3), melanoma differentiation-associated 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I), have been shown to be differentially expressed in neuroblastoma (NB) and promote dsRNA poly (I:C)-induced NB suppression in vitro and in vivo. However, the role of another important innate immune cytosolic sensor, laboratory of genetics and physiology 2 (LGP2), in the cancer behavior of NB remains unclear. Here, we demonstrated that the expression levels of LGP2 were either low or undetectable in all NB cell lines tested with or without MYCN amplification. LGP2 expression levels were significantly increased only in NB cells without MYCN amplification, including SK-N-AS and SK-N-FI after poly (I:C) treatment in vitro and in mouse xenograft models. Ectopic expression of LGP2 in NB cells significantly enhanced poly (I:C)-induced NB cell death associated with downregulation of MDA5, RIG-I, MAVS and Bcl-2, as well as upregulation of Noxa and tBid. By immunofluorescence analyses, LGP2 localized mainly in the cytoplasm of NB cells after poly (I:C) treatment. In human NB tissue samples, cytoplasmic LGP2 expression was positively correlated with histological differentiation and inversely correlated with MYCN amplification. Positive cytoplasmic LGP2 expression in tumor tissues could predict a favorable outcome in NB patients independent of other prognostic factors. In short, LGP2 was effective in promoting poly (I:C)-induced NB suppression and cytoplasmic LGP2 can serve as an independent favorable prognostic factor in NB patients.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) in fish: current knowledge and future perspectives.

Retinoic acid-inducible gene I (RIG-I) -like receptors (RLRs) are found conservatively present in teleost fish. All three members, RIG-I, MDA5 and LGP2, together with the downstream molecules such as MITA, TRAF3 and TBK1, have been identified in a range of fish species. However, it is unexpected that RIG-I has not been reported in fish of Acanthopterygii, and it would be important to clarify the presence and role of the RIG-I gene in a broad range of taxa in Teleostei. RLRs in fish can be induced in vivo and in vitro by viral pathogens as well as synthetic dsRNA, poly(I:C), leading to the production of type I interferons (IFNs) and the expression of IFN-stimulated genes (ISGs). Bacterial pathogens, such as Edwardsiella tarda, and their components, such as lipopolysaccharide are also found to induce the expression of RLRs, and whether such induction was mediated through the direct recognition by RLRs or through crosstalk with other pattern recognition receptors recognizing directly bacterial pathogen-associated molecular patterns awaits to be investigated. On the other hand, RLR-activated type I IFN production can be negatively regulated in fish by molecules, such as TBK-1-like protein and IRF10, which are found to negatively regulate RIG-I and MAVS-activated type I IFN production, and to block MITA or bind ISRE motifs, respectively. It is considered that the evolutionary occurrence of RLRs in fish, and their recognized ligands, especially those from their fish pathogens, as well as the mechanisms involved in the RLR signalling pathways, are of significant interest for further investigation.

PACT is required for MDA5-mediated immunoresponses triggered by Cardiovirus infection via interaction with LGP2.

Laboratory of genetics and physiology 2 (LGP2) and melanoma differentiation-associated gene 5 (MDA5) cooperatively detect viral RNA in the cytoplasm of Cardiovirus-infected cells and activate innate immune responses. Here, we evaluated whether the double-stranded RNA-binding protein PACT plays a role in this anti-viral response to further elucidate the mechanism. Immunoprecipitation experiments demonstrated that PACT interacts with LGP2 and that this interaction is enhanced by encephalomyocarditis virus (EMCV) infection. In vitro interaction analyses using purified recombinant proteins confirmed that the single-stranded Theiler's murine encephalitis virus genome enhanced the interaction between LGP2 and PACT. Small interfering RNA knockdown experiments further indicated that PACT is required for Cardiovirus-triggered interferon responses. To support this functional interaction with LGP2, overexpressed PACT was shown to enhance EMCV-triggered interferon promoter activity only when LGP2 and MDA5 were co-expressed but not when MDA5 is expressed alone. Together, our findings indicate a possible role of PACT in regulating the Cardiovirus-triggered immune responses mediated by MDA5 and LGP2, which opens the door to novel therapeutic strategies in interferon-related autoimmune diseases and cancer.