The Experts below are selected from a list of 1263 Experts worldwide ranked by ideXlab platform
Curt M Horvath - One of the best experts on this subject based on the ideXlab platform.
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RNA sensor LGP2 inhibits TRAF ubiquitin ligase to negatively regulate innate immune signaling.
EMBO reports, 2018Co-Authors: Jean Patrick Parisien, Annie M. Bruns, Kenny R Rodriguez, Jessica J. Lenoir, Roli Mandhana, Kenin Qian, Curt M HorvathAbstract:Abstract 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‐1β, 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.
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The TAR-RNA binding protein is required for immunoresponses triggered by Cardiovirus infection
Biochemical and biophysical research communications, 2016Co-Authors: Akihiko Komuro, Glen N Barber, Yuya Homma, Takaharu Negoro, Curt M HorvathAbstract:LGP2 and MDA5 cooperate to detect viral RNA in the cytoplasm of Picornavirus-infected cells and activate innate immune responses. To further define regulatory components of RNA recognition by LGP2/MDA5, a yeast two-hybrid screen was used to identify LGP2-interacting proteins. The screening has identified the TAR-RNA binding protein (TRBP), which is known to be an essential factor for RNA interference (RNAi). Immuno-precipitation experiments demonstrated that TRBP interacted specifically with LGP2 but not with related RIG-I-like receptors, RIG-I or MDA5. siRNA knockdown experiments indicate that TRBP is important for Cardiovirus-triggered interferon responses, but TRBP is not involved in Sendai virus-triggered interferon response that is mediated mainly by RIG-I. To support functional interaction with LGP2, overexpressed TRBP increased Cardiovirus-triggered interferon promoter activity only when LGP2 and MDA5 are co-expressed but not MDA5 alone. Together, our findings illustrate a possible connection between an RNAi-regulatory factor and antiviral RNA recognition that is specifically required for a branch of the virus induced innate immune response.
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LGP2 synergy with mda5 in rlr mediated rna recognition and antiviral signaling
Cytokine, 2015Co-Authors: Annie M. Bruns, Curt M HorvathAbstract:Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling. The RIG-I-like receptor (RLR) proteins are expressed in the cytoplasm of nearly all cells and specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RLR family is composed of three homologous proteins, RIG-I, MDA5, and LGP2. All RLRs have the ability to detect virus-derived dsRNA and hydrolyze ATP, but display individual differences in enzymatic activity, intrinsic ability to recognize RNA, and mechanisms of activation. Emerging evidence suggests that MDA5 and RIG-I utilize distinct mechanisms to form oligomeric complexes along dsRNA. Aligning of their signaling domains creates a platform capable of propagating and amplifying antiviral signaling responses. LGP2 with intact ATP hydrolysis is critical for the MDA5-mediated antiviral response, but LGP2 lacks the domains essential for activation of antiviral signaling, leaving the role of LGP2 in antiviral signaling unclear. Recent studies revealed a mechanistic basis of synergy between LGP2 and MDA5 leading to enhanced antiviral signaling. This review briefly summarizes the RLR system, and focuses on the relationship between LGP2 and MDA5, describing in detail how these two proteins work together to detect foreign RNA and generate a fully functional antiviral response.
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LGP2 synergy with mda5 in rlr mediated rna recognition and antiviral signaling
Cytokine, 2015Co-Authors: Annie M. Bruns, Curt M HorvathAbstract:Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling. The RIG-I-like receptor (RLR) proteins are expressed in the cytoplasm of nearly all cells and specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RLR family is composed of three homologous proteins, RIG-I, MDA5, and LGP2. All RLRs have the ability to detect virus-derived dsRNA and hydrolyze ATP, but display individual differences in enzymatic activity, intrinsic ability to recognize RNA, and mechanisms of activation. Emerging evidence suggests that MDA5 and RIG-I utilize distinct mechanisms to form oligomeric complexes along dsRNA. Aligning of their signaling domains creates a platform capable of propagating and amplifying antiviral signaling responses. LGP2 with intact ATP hydrolysis is critical for the MDA5-mediated antiviral response, but LGP2 lacks the domains essential for activation of antiviral signaling, leaving the role of LGP2 in antiviral signaling unclear. Recent studies revealed a mechanistic basis of synergy between LGP2 and MDA5 leading to enhanced antiviral signaling. This review briefly summarizes the RLR system, and focuses on the relationship between LGP2 and MDA5, describing in detail how these two proteins work together to detect foreign RNA and generate a fully functional antiviral response.
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ID: 82: The Innate Immune Sensor, LGP2, Interferes with TRAF Proteins to Prevent NFkB Activation
Cytokine, 2015Co-Authors: Curt M HorvathAbstract:The detection of RNA virus infections triggers the production type I interferon (IFN), and is a major component of the powerful antiviral system that directly interferes with virus replication and contributes to both innate and adaptive immune responses. Virus replication intermediates such as double stranded RNA (dsRNA) or RNAs with distinct non-self modifications are recognized in the cytoplasm by pattern recognition receptors including RIG-I, MDA5, and LGP2. These innate sensors recognize non-self RNAs via their C-terminal domains, and RIG-I and MDA5 use N-terminal CARDs to induce the activation of the downstream signaling scaffold, MAVS. MAVS coordinates the assembly of signaling machinery including TRAFs and IKK-family kinases that activate IRF3 and NFkB transcription factors. Available evidence suggests LGP2 acts as a concentration dependent switch to regulate RLR antiviral signaling. For positive actions, LGP2 uses its unique catalytic activity to enhance MDA5 RNA recognition, dsRNA filament assembly, and enable more efficient activation of MAVS and downstream signal transduction. The mechanism of LGP2 negative regulation have been investigated, and results indicate that LGP2-mediated inhibition occurs downstream of RIG-I, MDA5, and MAVS. LGP2 inhibition does not require ATPase activity or RNA binding. Instead, results indicate that LGP2 targets TRAF signaling proteins, disengaging their ubiquitin ligase activity, and potently preventing NFkB activation in MAVS dependent and independent systems. Identification of TRAF proteins as inhibitory targets not only resolves the dual roles of LGP2 in antiviral signaling regulation, but also expands the potential impact of LGP2 negative regulation beyond innate antiviral signaling.
Takashi Fujita - One of the best experts on this subject based on the ideXlab platform.
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Viral RNA recognition by LGP2 and MDA5, and activation of signaling through step-by-step conformational changes.
Nucleic acids research, 2020Co-Authors: Ivana Duic, Hiroki Kato, Hisashi Tadakuma, Yoshie Harada, Ryo Yamaue, Katashi Deguchi, Yuki Suzuki, Shige H. Yoshimura, Kunio Takeyasu, Takashi FujitaAbstract:Cytoplasmic RIG-I-like receptor (RLR) proteins in mammalian cells recognize viral RNA and initiate an antiviral response that results in IFN-β induction. Melanoma differentiation-associated protein 5 (MDA5) forms fibers along viral dsRNA and propagates an antiviral response via a signaling domain, the tandem CARD. The most enigmatic RLR, laboratory of genetics and physiology (LGP2), lacks the signaling domain but functions in viral sensing through cooperation with MDA5. However, it remains unclear how LGP2 coordinates fiber formation and subsequent MDA5 activation. We utilized biochemical and biophysical approaches to observe fiber formation and the conformation of MDA5. LGP2 facilitated MDA5 fiber assembly. LGP2 was incorporated into the fibers with an average inter-molecular distance of 32 nm, suggesting the formation of hetero-oligomers with MDA5. Furthermore, limited protease digestion revealed that LGP2 induces significant conformational changes on MDA5, promoting exposure of its CARDs. Although the fibers were efficiently dissociated by ATP hydrolysis, MDA5 maintained its active conformation to participate in downstream signaling. Our study demonstrated the coordinated actions of LGP2 and MDA5, where LGP2 acts as an MDA5 nucleator and requisite partner in the conversion of MDA5 to an active conformation. We revealed a mechanistic basis for LGP2-mediated regulation of MDA5 antiviral innate immune responses.
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ID: 70: A novel function of human Pumilio proteins in antiviral innate immunity
Cytokine, 2015Co-Authors: Ryo Narita, Kiyohiro Takahasi, Hiroki Kato, Mitsutoshi Yoneyama, Takashi FujitaAbstract: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-β 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.
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A Novel Function of Human Pumilio Proteins in Cytoplasmic Sensing of Viral Infection
PLoS Pathogens, 2014Co-Authors: Ryo Narita, Etsu Murakami, Emi Hirano, Kiyohiro Takahasi, Hiroki Kato, Seiji P. Yamamoto, Mitsutoshi Yoneyama, Takashi FujitaAbstract: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-β 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.
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LGP2 is a positive regulator of rig i and mda5 mediated antiviral responses
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Takashi Satoh, Hiroki Kato, Tohru Tsujimura, Kazufumi Matsushita, Mitsutoshi Yoneyama, Yutaro Kumagai, Shintaro Sato, Shizuo Akira, Takashi Fujita, Osamu TakeuchiAbstract: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-β 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-β 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.
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LGP2 is a positive regulator of rig i and mda5 mediated antiviral responses
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Takashi Satoh, Hiroki Kato, Tohru Tsujimura, Kazufumi Matsushita, Mitsutoshi Yoneyama, Yutaro Kumagai, Shintaro Sato, Shizuo Akira, Takashi Fujita, Osamu TakeuchiAbstract: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.
Mitsutoshi Yoneyama - One of the best experts on this subject based on the ideXlab platform.
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LGP2 virus sensor enhances apoptosis by upregulating apoptosis regulatory genes through TRBP-bound miRNAs during viral infection
Nucleic acids research, 2019Co-Authors: Tomoko Takahashi, Mitsutoshi Yoneyama, Yuko Nakano, Koji Onomoto, Kumiko Ui-teiAbstract: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.
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LGP2 virus sensor regulates gene expression network mediated by TRBP-bound microRNAs.
Nucleic acids research, 2018Co-Authors: Tomoko Takahashi, Mitsutoshi Yoneyama, Yuko Nakano, Koji Onomoto, Fuminori Murakami, Chiaki Komori, Yutaka Suzuki, Kumiko Ui-teiAbstract: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.
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Virus Sensor RIG-I Represses RNA Interference by Interacting with TRBP through LGP2 in Mammalian Cells
MDPI AG, 2018Co-Authors: Tomoko Takahashi, Mitsutoshi Yoneyama, Yuko Nakano, Koji Onomoto, Kumiko Ui-teiAbstract:Exogenous double-stranded RNAs (dsRNAs) similar to viral RNAs induce antiviral RNA silencing or RNA interference (RNAi) in plants or invertebrates, whereas interferon (IFN) response is induced through activation of virus sensor proteins including Toll like receptor 3 (TLR3) or retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) in mammalian cells. Both RNA silencing and IFN response are triggered by dsRNAs. However, the relationship between these two pathways has remained unclear. Laboratory of genetics and physiology 2 (LGP2) is one of the RLRs, but its function has remained unclear. Recently, we reported that LGP2 regulates endogenous microRNA-mediated RNA silencing by interacting with an RNA silencing enhancer, TAR-RNA binding protein (TRBP). Here, we investigated the contribution of other RLRs, RIG-I and melanoma-differentiation-associated gene 5 (MDA5), in the regulation of RNA silencing. We found that RIG-I, but not MDA5, also represses short hairpin RNA (shRNA)-induced RNAi by type-I IFN. Our finding suggests that RIG-I, but not MDA5, interacts with TRBP indirectly through LGP2 to function as an RNAi modulator in mammalian cells
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ID: 70: A novel function of human Pumilio proteins in antiviral innate immunity
Cytokine, 2015Co-Authors: Ryo Narita, Kiyohiro Takahasi, Hiroki Kato, Mitsutoshi Yoneyama, Takashi FujitaAbstract: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-β 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.
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A Novel Function of Human Pumilio Proteins in Cytoplasmic Sensing of Viral Infection
PLoS Pathogens, 2014Co-Authors: Ryo Narita, Etsu Murakami, Emi Hirano, Kiyohiro Takahasi, Hiroki Kato, Seiji P. Yamamoto, Mitsutoshi Yoneyama, Takashi FujitaAbstract: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-β 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.
Akihiko Komuro - One of the best experts on this subject based on the ideXlab platform.
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PACT is required for MDA5-mediated immunoresponses triggered by Cardiovirus infection via interaction with LGP2
Biochemical and Biophysical Research Communications, 2017Co-Authors: Masahiko Miyamoto, Akihiko KomuroAbstract:Abstract 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.
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The TAR-RNA binding protein is required for immunoresponses triggered by Cardiovirus infection
Biochemical and biophysical research communications, 2016Co-Authors: Akihiko Komuro, Glen N Barber, Yuya Homma, Takaharu Negoro, Curt M HorvathAbstract:LGP2 and MDA5 cooperate to detect viral RNA in the cytoplasm of Picornavirus-infected cells and activate innate immune responses. To further define regulatory components of RNA recognition by LGP2/MDA5, a yeast two-hybrid screen was used to identify LGP2-interacting proteins. The screening has identified the TAR-RNA binding protein (TRBP), which is known to be an essential factor for RNA interference (RNAi). Immuno-precipitation experiments demonstrated that TRBP interacted specifically with LGP2 but not with related RIG-I-like receptors, RIG-I or MDA5. siRNA knockdown experiments indicate that TRBP is important for Cardiovirus-triggered interferon responses, but TRBP is not involved in Sendai virus-triggered interferon response that is mediated mainly by RIG-I. To support functional interaction with LGP2, overexpressed TRBP increased Cardiovirus-triggered interferon promoter activity only when LGP2 and MDA5 are co-expressed but not MDA5 alone. Together, our findings illustrate a possible connection between an RNAi-regulatory factor and antiviral RNA recognition that is specifically required for a branch of the virus induced innate immune response.
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impaired cellular responses to cytosolic dna or infection with listeria monocytogenes and vaccinia virus in the absence of the murine LGP2 protein
PLOS ONE, 2011Co-Authors: Darja Pollpeter, Akihiko Komuro, Glen N Barber, Curt M HorvathAbstract:Innate immune signaling is crucial for detection of and the initial response to microbial pathogens. Evidence is provided indicating that LGP2, a DEXH box domain protein related to the RNA recognition receptors RIG-I and MDA5, participates in the cellular response to cytosolic double-stranded DNA (dsDNA). Analysis of embryonic fibroblasts and macrophages from mice harboring targeted disruption in the LGP2 gene reveals that LGP2 can act as a positive regulator of type I IFN and anti-microbial gene expression in response to transfected dsDNA. Results indicate that infection of LGP2-deficient mice with an intracellular bacterial pathogen, Listeria monocytogenes, leads to reduced levels of type I IFN and IL12, and allows increased bacterial growth in infected animals, resulting in greater colonization of both spleen and liver. Responses to infection with vaccinia virus, a dsDNA virus, are also suppressed in cells lacking LGP2, reinforcing the ability of LGP2 to act as a positive regulator of antiviral signaling. In vitro mechanistic studies indicate that purified LGP2 protein does not bind DNA but instead mediates these responses indirectly. Data suggest that LGP2 may be acting downstream of the intracellular RNA polymerase III pathway to activate anti-microbial signaling. Together, these findings demonstrate a regulatory role for LGP2 in the response to cytosolic DNA, an intracellular bacterial pathogen, and a DNA virus, and provide a plausible mechanistic hypothesis as the basis for this activity.
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a shared interface mediates paramyxovirus interference with antiviral rna helicases mda5 and LGP2
Journal of Virology, 2009Co-Authors: Jean Patrick Parisien, Akihiko Komuro, Glen N Barber, Darja Bamming, Aparna Ramachandran, Jason J Rodriguez, Robert D Wojahn, Curt M HorvathAbstract:Diverse members of the Paramyxovirus family of negative-strand RNA viruses effectively suppress host innate immune responses through the actions of their V proteins. The V protein mediates interference with the interferon regulatory RNA helicase MDA5 to avoid cellular antiviral responses. Analysis of the interaction interface revealed the MDA5 helicase C domain as necessary and sufficient for association with V proteins from human parainfluenza virus type 2, parainfluenza virus type 5, measles virus, mumps virus, Hendra virus, and Nipah virus. The identified approximately 130-residue region is highly homologous between MDA5 and the related antiviral helicase LGP2, but not RIG-I. Results indicate that the paramyxovirus V proteins can also associate with LGP2. The V protein interaction was found to disrupt ATP hydrolysis mediated by both MDA5 and LGP2. These findings provide a potential mechanistic basis for V protein-mediated helicase interference and identify LGP2 as a second cellular RNA helicase targeted by paramyxovirus V proteins.
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a shared interface mediates paramyxovirus interference with antiviral rna helicases mda5 and LGP2
Journal of Virology, 2009Co-Authors: Jean Patrick Parisien, Akihiko Komuro, Glen N Barber, Darja Bamming, Aparna Ramachandran, Jason J Rodriguez, Robert D Wojahn, Curt M HorvathAbstract:Detection of pathogens by the mammalian innate immune system is mediated by pattern recognition receptors. For viruses, nucleic acids are often the trigger for innate responses that culminate in antiviral gene expression, including the production of type I interferon (IFN-α/β). Foreign nucleic acids outside the cell can be recognized by transmembrane Toll-like receptors at the cell surface or in the lumen of endocytic vesicles (9). Intracellular nucleic acids are recognized by cytoplasmic receptor proteins (24). In both cases, receptor binding to the nucleic acid ligand triggers a signal transduction cascade that activates immediate transcriptional responses, including the induction of the antiviral cytokines in the IFN family. An important class of receptors for the detection of cytosolic nonself RNAs is represented by the proteins encoded by retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) (24). These proteins share functional domains, including an amino-terminal protein interaction motif that functions in signal transduction and is homologous to the caspase activation and recruitment domain (CARD) (8) and a C-terminal DECH-box family RNA helicase domain. The helicase domains share approximately 30 to 40% overall sequence identity. Experimental and structural studies have demonstrated a third functional region at the extreme C terminus of RIG-I that forms a zinc-mediated fold and functions as a regulatory domain for the recognition and discrimination of RNA 5′ ends (6, 20). Current evidence supports a model in which binding to RNA ligand induces a change in accessibility to the CARD, allowing it to interact with downstream signaling proteins, including the mitochondrial resident IPS-1 (also identified as MAVS, VISA, and Cardiff [12, 15, 21, 28]). This association coordinates a serine kinase-mediated cascade that activates latent transcription factors, including NF-κB and IFN regulatory factor 3, culminating in the expression of IFN-β and a number of other crucial antiviral effector genes (8). A third helicase protein, LGP2, resembles RIG-I and MDA5 in the helicase domain, also exhibiting 30 to 40% sequence identity. This overall level of similarity may underestimate the relatedness of these proteins, as they are more identical in their key helicase sequence motifs and encompassing domains (2). Significantly, LGP2 lacks the N-terminal CARD homology region. As a result, LGP2 expression is not typically associated with the ability to directly activate downstream signaling. Ectopic expression of LGP2 interferes with double-stranded RNA (dsRNA) and virus-induced antiviral signaling, while reduction of LGP2 expression by RNA interference results in enhanced IFN-β synthesis and antiviral responses. Expression of LGP2, like that of RIG-I and MDA5, is induced by virus infection, nucleic acid transfection, and IFN stimulation, suggesting it has the properties of a negative feedback inhibitor (14, 19, 20, 29). The importance of these RNA helicase proteins in antiviral responses is validated by the phenotypes of mice harboring targeted disruptions in these genes (9). Deficiency in RIG-I leads to a widespread enhancement in replication of many RNA virus types, due to suppressed IFN biosynthesis and antiviral responses (11). Cells deficient in RIG-I exhibit a general defect in the ability to respond to foreign dsRNAs or single-stranded RNAs (ssRNAs) bearing phosphorylated 5′ ends. MDA5 deficiency showed a more specific phenotype, resulting in increased susceptibility to picornavirus infection and insensitivity to the synthetic dsRNA analog poly(I:C) (7). LGP2 deficiency leads to a more complex phenotype (27). LGP2-deficient mice are more sensitive to IFN induction by cytosolic poly(I:C), an MDA5 ligand, and more resistant to vesicular stomatitis virus, a virus that triggers RIG-I signaling. Negative regulation of the IFN response remains intact overall, indicating that LGP2 may not be the primary negative regulator of type I IFN production. However, LGP2 deficiency results in a suppressed IFN response to infection with encephalomyocarditis virus, a picornavirus that has been demonstrated to trigger MDA5-mediated antiviral signaling. These data suggest that LGP2 executes both negative and positive regulatory functions related to RIG-I and MDA5 signaling. The disparate effects of LGP2 deficiency are difficult to interpret without a better mechanistic understanding of LGP2 functions in cellular innate antiviral immunity. Further affirmation of the antiviral role of the helicase proteins derives from the fact that individual viruses have evolved means to evade or disrupt their activity, either by direct interference or by antagonizing the signaling intermediates. The large Paramyxovirus family of negative-strand RNA viruses is well known to evade IFN antiviral responses. Most viruses within this family encode a protein, called V, that is essential for IFN signaling evasion. In addition to suppressing the activity of signal transducer and activator of transcription (STAT) proteins to compromise IFN signal transduction, paramyxovirus V proteins can also limit the induction of IFN gene expression by interfering with the MDA5 protein (1, 3). Associations between the V protein C-terminal domain (CTD), a 60- to 70-residue zinc binding fold that is the conserved hallmark of V proteins, and the MDA5 helicase domain prevents signal transduction downstream of poly(I:C), a synthetic RNA ligand that is a known MDA5 activator (1, 3). This inhibition was observed for all tested V proteins of the large Paramyxovirus family (3). Despite the potential importance of the V protein-MDA5 interface as an antiviral target, the mechanistic basis and consequences for MDA5 interference by V proteins are poorly understood and difficult to reconcile with the lack of phenotype in MDA5-deficient mice. Recent evidence indicates that MDA5 is involved in the detection of Sendai virus-defective interfering (DI) genomes, providing plausible biological relevance to the observed V protein interference that may not have been apparent from gene disruption studies (31). Identification of the MDA5 target recognized by paramyxovirus V proteins led to the discovery that LGP2 is also a target for V protein antagonism. Results demonstrate that the helicase C domain of both proteins functions as a V protein binding region common to both MDA5 and LGP2 but absent in RIG-I. We demonstrate that interaction with the V protein interferes with the catalytic activity of both target helicases, providing a biochemical basis for V protein helicase antagonism.
Kenny R Rodriguez - One of the best experts on this subject based on the ideXlab platform.
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RNA sensor LGP2 inhibits TRAF ubiquitin ligase to negatively regulate innate immune signaling.
EMBO reports, 2018Co-Authors: Jean Patrick Parisien, Annie M. Bruns, Kenny R Rodriguez, Jessica J. Lenoir, Roli Mandhana, Kenin Qian, Curt M HorvathAbstract:Abstract 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‐1β, 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.
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paramyxovirus v protein interaction with the antiviral sensor LGP2 disrupts mda5 signaling enhancement but is not relevant to LGP2 mediated rlr signaling inhibition
Journal of Virology, 2014Co-Authors: Kenny R Rodriguez, Curt M HorvathAbstract:ABSTRACT The interferon antiviral system is a primary barrier to virus replication triggered upon recognition of nonself RNAs by the cytoplasmic sensors encoded by retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology gene 2 (LGP2). Paramyxovirus V proteins are interferon antagonists that can selectively interact with MDA5 and LGP2 through contact with a discrete helicase domain region. Interaction with MDA5, an activator of antiviral signaling, disrupts interferon gene expression and antiviral responses. LGP2 has more diverse reported roles as both a coactivator of MDA5 and a negative regulator of both RIG-I and MDA5. This functional dichotomy, along with the concurrent interference with both cellular targets, has made it difficult to assess the unique consequences of V protein interaction with LGP2. To directly evaluate the impact of LGP2 interference, MDA5 and LGP2 variants unable to be recognized by measles virus and parainfluenza virus 5 (PIV5) V proteins were tested in signaling assays. Results indicate that interaction with LGP2 specifically prevents coactivation of MDA5 signaling and that LGP29s negative regulatory capacity was not affected. V proteins only partially antagonize RIG-I at high concentrations, and their expression had no additive effects on LGP2-mediated negative regulation. However, conversion of RIG-I to a direct V protein target was accomplished by only two amino acid substitutions that allowed both V protein interaction and efficient interference. These results clarify the unique consequences of MDA5 and LGP2 interference by paramyxovirus V proteins and help resolve the distinct roles of LGP2 in both activation and inhibition of antiviral signal transduction. IMPORTANCE Paramyxovirus V proteins interact with two innate immune receptors, MDA5 and LGP2, but not RIG-I. V proteins prevent MDA5 from signaling to the beta interferon promoter, but the consequences of LGP2 targeting are poorly understood. As the V protein targets MDA5 and LGP2 simultaneously, and LGP2 is both a positive and negative regulator of both MDA5 and RIG-I, it has been difficult to evaluate the specific advantages conferred by LGP2 targeting. Experiments with V-insensitive proteins revealed that the primary outcome of LGP2 interference is suppression of its ability to synergize with MDA5. LGP29s negative regulation of MDA5 and RIG-I remains intact irrespective of V protein interaction. Complementary experiments demonstrate that RIG-I can be converted to V protein sensitivity by two amino acid substitutions. These findings clarify the functions of LGP2 as a positive regulator of MDA5 signaling, demonstrate the basis for V-mediated LGP2 targeting, and broaden our understanding of paramyxovirus-host interactions.
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paramyxovirus v protein interaction with the antiviral sensor LGP2 disrupts mda5 signaling enhancement but is not relevant to LGP2 mediated rlr signaling inhibition
Journal of Virology, 2014Co-Authors: Kenny R Rodriguez, Curt M HorvathAbstract:ABSTRACT The interferon antiviral system is a primary barrier to virus replication triggered upon recognition of nonself RNAs by the cytoplasmic sensors encoded by retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology gene 2 (LGP2). Paramyxovirus V proteins are interferon antagonists that can selectively interact with MDA5 and LGP2 through contact with a discrete helicase domain region. Interaction with MDA5, an activator of antiviral signaling, disrupts interferon gene expression and antiviral responses. LGP2 has more diverse reported roles as both a coactivator of MDA5 and a negative regulator of both RIG-I and MDA5. This functional dichotomy, along with the concurrent interference with both cellular targets, has made it difficult to assess the unique consequences of V protein interaction with LGP2. To directly evaluate the impact of LGP2 interference, MDA5 and LGP2 variants unable to be recognized by measles virus and parainfluenza virus 5 (PIV5) V proteins were tested in signaling assays. Results indicate that interaction with LGP2 specifically prevents coactivation of MDA5 signaling and that LGP29s negative regulatory capacity was not affected. V proteins only partially antagonize RIG-I at high concentrations, and their expression had no additive effects on LGP2-mediated negative regulation. However, conversion of RIG-I to a direct V protein target was accomplished by only two amino acid substitutions that allowed both V protein interaction and efficient interference. These results clarify the unique consequences of MDA5 and LGP2 interference by paramyxovirus V proteins and help resolve the distinct roles of LGP2 in both activation and inhibition of antiviral signal transduction. IMPORTANCE Paramyxovirus V proteins interact with two innate immune receptors, MDA5 and LGP2, but not RIG-I. V proteins prevent MDA5 from signaling to the beta interferon promoter, but the consequences of LGP2 targeting are poorly understood. As the V protein targets MDA5 and LGP2 simultaneously, and LGP2 is both a positive and negative regulator of both MDA5 and RIG-I, it has been difficult to evaluate the specific advantages conferred by LGP2 targeting. Experiments with V-insensitive proteins revealed that the primary outcome of LGP2 interference is suppression of its ability to synergize with MDA5. LGP29s negative regulation of MDA5 and RIG-I remains intact irrespective of V protein interaction. Complementary experiments demonstrate that RIG-I can be converted to V protein sensitivity by two amino acid substitutions. These findings clarify the functions of LGP2 as a positive regulator of MDA5 signaling, demonstrate the basis for V-mediated LGP2 targeting, and broaden our understanding of paramyxovirus-host interactions.
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amino acid requirements for mda5 and LGP2 recognition by paramyxovirus v proteins a single arginine distinguishes mda5 from rig i
Journal of Virology, 2013Co-Authors: Kenny R Rodriguez, Curt M HorvathAbstract:Paramyxovirus V proteins bind to MDA5 (melanoma differentiation-associated gene 5) and LGP2 (laboratory of genetics and physiology gene 2) but not RIG-I (retinoic acid-inducible gene I). The results demonstrate MDA5 R806 is essential for inhibition by diverse V proteins. Complementary substitution for the analogous RIG-I L714 confers V protein recognition. The analogous LGP2 R455 is required for recognition by measles V protein, but not other V proteins. These findings indicate that paramyxoviruses use a single amino acid to distinguish MDA5 from RIG-I and have evolved distinct contact sites for LGP2 interference.