Laboratory Of Genetics And Physiology 2 Research Articles

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

Hepatitis A virus (HAV) infections are considered not to trigger innate immunity invivo, 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 invivo and invitro. 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-β (TRIF) and mitochondrial antiviral-signalling protein (MAVS) was analysed by transient and stable expression of HAV and HCV proteases and virus infection. 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. 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. 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 invitro and invivo, 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.

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.

Efficient Dicer processing of virus-derived double-stranded RNAs and its modulation by RIG-I-like receptor LGP2.

The interferon-regulated antiviral responses are essential for the induction of both innate and adaptive immunity in mammals. Production of virus-derived small-interfering RNAs (vsiRNAs) to restrict virus infection by RNA interference (RNAi) is a recently identified mammalian immune response to several RNA viruses, which cause important human diseases such as influenza and Zika virus. However, little is known about Dicer processing of viral double-stranded RNA replicative intermediates (dsRNA-vRIs) in mammalian somatic cells. Here we show that infected somatic cells produced more influenza vsiRNAs than cellular microRNAs when both were produced by human Dicer expressed de novo, indicating that dsRNA-vRIs are not poor Dicer substrates as previously proposed according to in vitro Dicer processing of synthetic long dsRNA. We report the first evidence both for canonical vsiRNA production during wild-type Nodamura virus infection and direct vsiRNA sequestration by its RNAi suppressor protein B2 in two strains of suckling mice. Moreover, Sindbis virus (SINV) accumulation in vivo was decreased by prior production of SINV-targeting vsiRNAs triggered by infection and increased by heterologous expression of B2 in cis from SINV genome, indicating an antiviral function for the induced RNAi response. These findings reveal that unlike artificial long dsRNA, dsRNA-vRIs made during authentic infection of mature somatic cells are efficiently processed by Dicer into vsiRNAs to direct antiviral RNAi. Interestingly, Dicer processing of dsRNA-vRIs into vsiRNAs was inhibited by LGP2 (laboratory of genetics and physiology 2), which was encoded by an interferon-stimulated gene (ISG) shown recently to inhibit Dicer processing of artificial long dsRNA in cell culture. Our work thus further suggests negative modulation of antiviral RNAi by a known ISG from the interferon response.

Innate immune sensor LGP2 is cleaved by the Leader protease of foot-and-mouth disease virus.

The RNA helicase LGP2 (Laboratory of Genetics and Physiology 2) is a non-signaling member of the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), whose pivotal role on innate immune responses against RNA viruses is being increasingly uncovered. LGP2 is known to work in synergy with melanoma differentiation-associated gene 5 (MDA5) to promote the antiviral response induced by picornavirus infection. Here, we describe the activity of the foot-and-mouth disease virus (FMDV) Leader protease (Lpro) targeting LGP2 for cleavage. When LGP2 and Lpro were co-expressed, cleavage products were observed in an Lpro dose-dependent manner while co-expression with a catalytically inactive Lpro mutant had no effect on LGP2 levels or pattern. We further show that Lpro localizes and immunoprecipitates with LGP2 in transfected cells supporting their interaction within the cytoplasm. Evidence of LGP2 proteolysis was also detected during FMDV infection. Moreover, the inhibitory effect of LGP2 overexpression on FMDV growth observed was reverted when Lpro was co-expressed, concomitant with lower levels of IFN-β mRNA and antiviral activity in those cells. The Lpro target site in LGP2 was identified as an RGRAR sequence in a conserved helicase motif whose replacement to EGEAE abrogated LGP2 cleavage by Lpro. Taken together, these data suggest that LGP2 cleavage by the Leader protease of aphthoviruses may represent a novel antagonistic mechanism for immune evasion.

Foot-and-mouth disease virus infection inhibits LGP2 protein expression to exaggerate inflammatory response and promote viral replication

The role of the innate immune protein LGP2 (laboratory of genetics and physiology 2) in FMDV-infected cells remains unknown. Here, we demonstrate the antiviral role of LGP2 during FMDV infection. FMDV infection triggered LGP2 mRNA expression but reduced protein expression. Overexpression of LGP2 suppressed FMDV replication, and the inflammatory response was significantly inhibited by LGP2 in virus-infected cells. The N-terminal DExDc and the C-terminal regulatory domain regions of LGP2 were essential for LGP2-mediated antiviral activity against FMDV. Disruption of RNA recognition by LGP2 is suggested to abolish completely LGP2-mediated antiviral activity against FMDV. FMDV leader protein (Lpro), as well as the 3Cpro and 2B proteins were determined to possess the ability to induce reduction of LGP2 protein expression. 2B-induced reduction of LGP2 was independent of cleavage of eukaryotic translation initiation factor 4 gamma; and the proteasomes, lysosomes or caspase-dependent pathways were not involved in this process. The C-terminal amino acids of 101–154 were essential for 2B-induced reduction of LGP2 and upregulation of inflammatory response. Direct interaction was demonstrated between LGP2 and 2B. Our results describe the antiviral role of LGP2 against FMDV and a novel antagonistic mechanism of FMDV that is mediated by 2B protein.

Identification, ontogeny and expression analysis of a novel laboratory of genetics and physiology 2 (LGP2) transcript in Asian seabass, Lates calcarifer

LGP2 (laboratory of genetics and physiology 2) is an important member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), which plays a significant role in antiviral innate immunity. In this study, we have cloned the full-length cDNA sequence of LGP2 from Asian seabass, Lates calcarifer (AsLGP2). The complete AsLGP2 cDNA sequence consisted of 2586 nucleotides encoding a putative protein of 681 amino acids with a molecular mass of 77.6 kDa. From the AsLGP2 protein, four different conserved domains were predicted: a DExDc (DEAD/DEAH box helicase domain), a bacterial type III restriction enzyme domain (RES III), a HELICc (Helicase superfamily c-terminal domain and a RIG-I_C-RD (RIG-I C-terminal regulatory domain). The transcript of AsLGP2 could be detected in all the 11 tissues tested in healthy animals with high expression noticed in tissues facing external environment such as gill, hindgut and skin. The ontogenic expression profile of AsLGP2 implies a possible maternal transfer of this gene as it has been detected in all early embryonic developmental stages along with unfertilized eggs. Viral analogue, poly I:C, injection resulted in rapid up-regulated expression in different tissues with the highest modulation of expression observed in kidney followed by liver and gill. A rapid response of AsLGP2 expression was also observed in the different tissues of Vibrio alginolyticus-injected L. calcarifer, while significant change in expression was noticed following Staphylococcus aureus infection. Similarly, exposure to different pathogen-mimicking microbial analogues such as poly I:C, LPS and PGN resulted in enhanced expression of AsLGP2 in SISK cell-line. Taking together, these observations suggest that AsLGP2 can act as both antiviral and antibacterial cytosolic receptor and may play a significant role in embryonic and larval development in marine euryhaline teleosts like Asian seabass.

Characterization and expression analysis of laboratory of genetics and physiology 2 gene in sea perch, Lateolabrax japonicus.

LGP2 (laboratory of genetics and physiology 2) as a key component of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), plays a predominant role in modulating RLRs-mediated cellular antiviral signaling during viral infection. In the present study, we cloned the LGP2 gene from the sea perch (Lateolabrax japonicus) (LjLGP2), an economically important farmed fish. The complete cDNA sequence of LjLGP2 was 2790 nt and encoded a polypeptide of 682 amino acids which contains four main structural domains: one DEAD/DEAH box helicase domain, one conserved restriction domain of bacterial type III restriction enzyme, one helicase superfamily c-terminal domain and one C-terminal domain of RIG-I, similar to most vertebrate LGP2. Subcellular localization analysis showed that LjLGP2 spanned the entire cytosol. The LjLGP2 mRNA was widespread expressed in the tested 10 tissues of healthy fish and significantly up-regulated post NNV infection. Furthermore, time course analysis showed that LjLGP2 transcripts significantly increased in the spleen, kidney and liver tissues after NNV infection. LjLGP2 mRNA expression was rapidly and significantly up-regulated in LJB cells after poly I:C stimulation and NNV infection. The present results suggest that LjLGP2 may be involved in recognization of NNV and play a role in antiviral innate immune against NNV in sea perch.

Characterization of the IFN pathway in the teleost fish gonad against vertically transmitted viral nervous necrosis virus.

One of the most powerful innate immune responses against viruses is mediated by type I IFN. In teleost fish, it is known that virus infection triggers the expression of ifn and many IFN-stimulated genes, but the viral RNA sensors and mediators leading to IFN production are scarcely known. Thus, we have searched for the presence of these genes in gilt-head sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax), and evaluated their expression after infection with viral nervous necrosis virus (VNNV) in the brain, the main viral target tissue, and the gonad, used to transmit the virus vertically. In sea bream, a fish species resistant to the VNNV strain used, we found an upregulation of the genes encoding MDA5 (melanoma differentiation-associated gene 5), TBK1 (TANK-binding kinase 1), IRF3 (IFN regulatory factor 3), IFN, Mx [myxovirus (influenza) resistance protein] and PKR (dsRNA-dependent protein kinase receptor) proteins in the brain, which were unaltered in the gonad and could favour the dissemination by gonad fluids or gametes. Strikingly, in European sea bass, a very susceptible species, we also identified, transcripts coding for LGP2 (Laboratory of Genetics and Physiology 2), MAVS (mitochondrial antiviral signalling), TRAF3 (TNF receptor-associated factor 3), TANK (TRAF family member-associated NFκB activator) and IRF7 (IFN regulatory factor 7), and found that all the genes analysed were upregulated in the gonad, but only mda5, lgp2, irf3, mx and pkr were upregulated in the brain. These findings supported the notion that the European sea bass brain innate immune response is unable to clear the virus and pointed to the importance of gonad immunity to control the dissemination of VNNV to the progeny--an aspect that is worth investigating in aquatic animals.

LGP2 plays extensive roles in modulating innate immune responses in Ctenopharyngodon idella kidney (CIK) cells

LGP2 (laboratory of genetics and physiology 2), RIG-I (retinoic acid inducible gene-I) and MDA5 (melanoma differentiation associated gene 5) constitute the RLR (RIG-I-like receptor) family. LGP2 plays a pivotal role in modulating signaling of RIG-I and MDA5 in innate immune responses. In this study, three representative overexpression vectors were constructed and transfected into C. idella kidney (CIK) cell line to research functional characterizations of CiLGP2 (C. idella LGP2). CiLGP2 overexpression led to the induction of CiRIG-I transcripts. After GCRV challenge, CiLGP2 enhanced CiMDA5 and CiIPS-1 to reinforce the immune response, however, impaired the expression of CiRIG-I. Meanwhile, antiviral activity assays showed that overexpression of CiLGP2 or its domains could inhibit GCRV replication and protect cells from death. Besides, CiLGP2 lingeringly induced CiRIG-I mRNA expression and inhibited CiMDA5 transcripts post poly(I:C) simulation. As a result, CiLGP2 suppressed the RLR-mediated signaling pathway against poly(I:C). Furthermore, CiLGP2 played active roles in RLR signaling response to bacterial PAMPs (LPS and PGN) stimulation. CiLGP2 altered the expression pattern of CiIPS-1 after LPS treatment, while it significantly enhanced the RLR signaling pathway against PGN stimulation. These results collectively suggested that CiLGP2 played a strikingly broad regulation in RLR mediated innate immune responses in C. idella, responding to not only the dsRNA virus or synthetic dsRNA but also bacterial PAMPs, which contribute to the understanding of C. idella LGP2 and RLR signaling pathways. In addition, these results lay a foundation for the further functional mechanism research of LGP2 in fishes.

Expression of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) in the human term placenta

Event Abstract Back to Event Expression of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) in the human term placenta Agnieszka Jabłońska1, Patrycja Suski1, Mirosława Studzińska1, Jarosław Kalinka2 and Edyta Paradowska1* 1 Institute of Medical Biology Polish Academy of Sciences, Laboratory of Molecular Virology and Biological Chemistry, Poland 2 Medical University of Lodz, Department of Perinatology, First Chair of Gynecology and Obstetrics, Poland Innate recognition of viruses is mediated by germline-encoded pattern recognition receptors (PRRs) that initiate signaling events, resulting in the production of type I interferon and proinflammatory cytokines. Double-stranded RNA and 5’triphosphate group of ssRNA generated during viral replication are detected by sensory molecules, named RIG-I-like receptors (RLRs). Three members of the RLRs family have been characterized so far, including RIG-I (retinoic acid-inducible gene-I), MDA5 (melanoma differentation-associated gene 5) and LGP2 (laboratory of genetics and physiology 2). RIG-I participates in the recognition of Paramyxoviruses, Rhabdoviruses, Flaviviruses and Orthomyxoviruses, whereas MDA-5 is essential for the recognition of Picornaviruses. In this study, we investigated the RLRs gene expression in fetal and maternal parts of the human placentas during viral infection. The explants of third-trimester chorionic villi and deciduas were exposed to infection with dsDNA viruses (human cytomegalovirus, HCMV; herpes simplex virus type 1, HSV1) or ssRNA virus (vesicular stomatitis virus, VSV). RIG-I, MDA5 and LGP2 mRNA levels were assessed by real-time PCR. Our results demonstrated that all RLRs were expressed in term human placentas with the highest level of LGP2. RIG-I and MDA5 (in fetal part) have been identified as the main mediators of VSV recognition, whereas RLRs were not involved in the identification of dsDNA viruses. The expression of LGP2 in HCMV-infected explants was decreased compared to mock-infected. The results clearly indicated that RLRs play essential role in RNA viruses’ recognition in term placenta. Acknowledgements Project financed by the European Regional Development Found under the Operational Programme Innovative Economy, Grant No. POIG.01.01.02-10-107/09. Keywords: human placenta, Virus infection, RLRs, RIG-I-like receptors, LGP2 Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Jabłońska A, Suski P, Studzińska M, Kalinka J and Paradowska E (2013). Expression of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) in the human term placenta. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00293 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 13 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Edyta Paradowska, Institute of Medical Biology Polish Academy of Sciences, Laboratory of Molecular Virology and Biological Chemistry, Lodz, 93-232, Poland, eparadowska@cbm.pan.pl Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Agnieszka Jabłońska Patrycja Suski Mirosława Studzińska Jarosław Kalinka Edyta Paradowska Google Agnieszka Jabłońska Patrycja Suski Mirosława Studzińska Jarosław Kalinka Edyta Paradowska Google Scholar Agnieszka Jabłońska Patrycja Suski Mirosława Studzińska Jarosław Kalinka Edyta Paradowska PubMed Agnieszka Jabłońska Patrycja Suski Mirosława Studzińska Jarosław Kalinka Edyta Paradowska Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

LGP2 Expression is Enhanced by Interferon Regulatory Factor 3 in Olive Flounder, Paralichthys olivaceus

In innate immunity, LGP2 (laboratory of genetics and physiology 2) plays a very important role in the production of type I interferon (IFN) through recognition of cytosolic viral RNA. Although viral infection or stimulation with double-strand RNA dramatically induces expression of the LGP2 gene, the underlying transcriptional mechanism has never been studied. Here, we cloned and characterized the 5′-upstream region (−1,337 bp) of the LGP2 gene in olive flounder (Paralichthys olivaceus). Numerous canonical motifs for IFN-regulatory factors (IRFs) were found in this region, and reporter assays identified a poly I:C-responsive promoter region (−506 to −398) that regulated LGP2 transcription. Transcriptional activity of the LGP2 promoter was strongly enhanced by IRF3, which bound to IRF3 motif #3 (−480). The LGP2 promoter was also responsive to viral infection in vitro. These results suggest that LGP2 transcriptional control is crucially involved to regulated by IRF3 function after viral infection or stimulation with poly I:C.

A Novel Dermato-Pulmonary Syndrome Associated With MDA-5 Antibodies

Melanoma differentiation-associated protein 5 (MDA-5) is a novel autoantibody frequently characterized by interstitial lung disease and a distinct cutaneous phenotype with palmar papules, ulceration, and rash. Virtually all patients have underlying dermatomyositis, but many lack the characteristic clinical myopathy associated with it. In the setting of amyopathic disease, the absence of clinically available biomarkers or clear pathologic diagnosis can complicate effective prognostic and therapeutic intervention. Until recently the presence of MDA-5 antibody associated dermato-pulmonary syndrome was described only in Asian populations. We present 2 cases of MDA-5-associated dermato-pulmonary syndrome and provide a comprehensive review of available literature.

Respiratory Syncytial Virus (RSV) Nonstructural Protein 1 (NS1) Regulates Host Defense by Modulating LGP2

RSV NS1 attenuates type-I IFN production by modulating RLRs (Rig-I like receptors), which comprise RIG-I (retinoic acid inducible gene), MAVS (mitochondrial antiviral signaling) and LGP2 (laboratory of genetics and physiology 2), however the precise mechanism of NS1 inhibition of the host's innate antiviral immune response is unclear. We have shown that NS1 localizes to the mitochondria following RSV infection, binds to MAVS and thereby inhibits MAVS-RIG-I interaction required for IFN production. Here we have found that NS1 also increases LGP2 expression and that LGP2 is required for NS1-mediated attenuation of IFN-β. Human alveolar epithelial A549 cells were infected with wt RSV or RSV with an NS1 deletion (ΔNS1) or transfected with plasmid encoding NS1. Interaction between MAVS and RIG-I and expression of LGP2 in the presence or absence of NS1 was determined by immunoprecipitation and western blot in both infection and transfection models. The role of LGP2 in the NS1-mediated immune response was investigated using siLGP2, qRT-PCR and immunoblot analysis in A549 cells and in a stable HEK-293 cell line for regulating expression of NS1. Results show that NS1 elevates the expression of LGP2 by immunoblot analyses. In a stable cell line, NS1 is shown to upregulate LGP2 expression by 16h post induction in a dose-dependent manner. Furthermore, downregulating LGP2 by the expression of siLGP2 prevents the NS1-induced attenuation of IFN-β. Together these results show that RSV-NS1 upregulates LGP2, which in turn downregulates IFN-β production, suggesting NS1 modulation of LGP2 provides an additional pathway for RSV regulation of host defense.

Regulation of RLR-mediated innate immune signaling – It is all about keeping the balance

The current view of cytoplasmic RNA-mediated innate immune signaling involves the differential activation of the RNA helicases retinoic acid-inducible gene 1 (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology-2 (LGP2) by distinct RNA viruses. RIG-I, MDA5 and LGP2 form the RIG-I like receptor family (RLR). Since the initial characterization of the RLRs rapid progress has been made in the understanding of the molecular mechanisms that upon virus infection lead to the activation of downstream signaling cascades and the subsequent induction of type I interferon (IFN) and proinflammatory cytokines by these receptors. However, antiviral responses must be tightly regulated in order to prevent uncontrolled production of type I IFN that might have deleterious effects on the host. Exploring the structural and molecular mechanisms that underlie RLR signaling thus was accompanied by the discovery of how RLR-dependent antiviral responses are modulated. This article summarizes the current understanding of endogenous regulation in RLR signaling by various intrinsic molecules that exert their regulatory function in both the steady state or upon viral infection by targeting multiple steps of the signaling cascade.

Evolutional conservation of cytosolic antiviral activity with type I interferon mediated by the retinoic acid-inducible gene I-like receptor pathway in teleosts (170.11)

Abstract The retinoic acid-inducible gene-I-like receptor (RLR) family, one of the pattern recognition receptors (PRRs), is important for the cytosolic immune response to recognize viral RNAs as pathogen-associated molecular patterns (PAMPs). RLR family members MDA5 (melanoma differentiation-associated gene 5) and LGP2 (laboratory of genetics and physiology 2) enhance gene expression of type-I interferon (IFN-I) and inflammatory cytokines through the IPS-1 (mitochondrial adaptor IFN-β promoter stimulator-1) cascade. In this study, MDA5, LGP2 and IPS-1 were cloned from the kidney of the Japanese flounder, Paralichthys olivaceus, and their phylogenetic analyses showed their conservation to those of higher vertebrates. MDA5 and LGP2 mRNA expression were strongly up-regulated by poly I:C stimulation or VHSV infection, whereas IPS-1 mRNA expression was not. To reveal the antiviral activity of these molecules, expression vectors were constructed and used in an antiviral assay against VHSV and HIRRV using the HINAE (Hirame natural embryonic) cell line. Viral replications were visibly repressed by overexpression of the MDA5, LGP2 and IPS-1 genes. The antiviral activities were attributed to the up-regulation of type I interferon (IFN) and IFN-inducible genes (Mx and ISG15). These results indicate that MDA5, LGP2 and IPS-1 are capable of causing the inhibition of viral replication through the induction of type I IFN and IFN-inducible genes.

Identification and expression profiling analysis of grass carp Ctenopharyngodon idella LGP2 cDNA

LGP2 (laboratory of genetics and physiology 2), a homologue of RIG-I (Retinoic acid inducible gene-I) and MDA5 (Melanoma differentiation associated gene 5) without the CARD (caspase activation and recruitment domain) required for signaling, plays a pivotal role in modulating signaling by RIG-I and MDA5 for interferon (IFN) synthesis. In this study, a novel LGP2 gene from grass carp Ctenopharyngodon idella (designated as CiLGP2) was isolated and characterized. The full-length cDNA of CiLGP2 was of 2920 bp with five instability motifs (ATTTA). The open reading frame was of 2043 bp and encoded a polypeptide of 680 amino acids, including five main overlapping structural domains: two DEXDc (DEAD/DEAH box helicase domain), one ResIII (conserved restriction domain of bacterial type III restriction enzyme), one HELICc (helicase superfamily c-terminal domain) and one RD (regulatory domain). There was one more α-helix in the RD, compared with that in human. The CiLGP2 mRNA was ubiquitous expression in the tested tissues, was high level in spleen, skin, heart and intestine tissues, and was up-regulated by grass carp reovirus (GCRV) injection by semi-quantitative RT-PCR (sqRT-PCR) assay. The CiLGP2 expression in spleen was significantly up-regulated at 12 h (14.5 folds, P < 0.05), reached the crest at 24 h (19.0 folds, P < 0.05), and then dropped a little at 48 h (10.4 folds) post-injection of GCRV and kept this level in the following test period ( P < 0.05). In liver, the temporal expression of CiLGP2 mRNA was significantly increased at 24 h (3.8 times, P < 0.05), reached peak at 48 h (10.7 times, P < 0.05), and then decreased a little bit at 72 h (5.8 times, P < 0.05) and kept this high level by the end of the test ( P < 0.05). These results collectively suggested that CiLGP2 was a novel member of RLR gene family, engaging in the early stage of antiviral innate immune defense in grass carp, and laid the foundation for the further mechanism research of LGP2 in fishes.

Solution Structures of Cytosolic RNA Sensor MDA5 and LGP2 C-terminal Domains: IDENTIFICATION OF THE RNA RECOGNITION LOOP IN RIG-I-LIKE RECEPTORS

The RIG-I like receptor (RLR) comprises three homologues: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2). Each RLR senses different viral infections by recognizing replicating viral RNA in the cytoplasm. The RLR contains a conserved C-terminal domain (CTD), which is responsible for the binding specificity to the viral RNAs, including double-stranded RNA (dsRNA) and 5'-triphosphated single-stranded RNA (5'ppp-ssRNA). Here, the solution structures of the MDA5 and LGP2 CTD domains were solved by NMR and compared with those of RIG-I CTD. The CTD domains each have a similar fold and a similar basic surface but there is the distinct structural feature of a RNA binding loop; The LGP2 and RIG-I CTD domains have a large basic surface, one bank of which is formed by the RNA binding loop. MDA5 also has a large basic surface that is extensively flat due to open conformation of the RNA binding loop. The NMR chemical shift perturbation study showed that dsRNA and 5'ppp-ssRNA are bound to the basic surface of LGP2 CTD, whereas dsRNA is bound to the basic surface of MDA5 CTD but much more weakly, indicating that the conformation of the RNA binding loop is responsible for the sensitivity to dsRNA and 5'ppp-ssRNA. Mutation study of the basic surface and the RNA binding loop supports the conclusion from the structure studies. Thus, the CTD is responsible for the binding affinity to the viral RNAs.