RESUMO
BACKGROUND@#The hepatitis B virus (HBV) vaccine has been efficiently used for decades. However, hepatocellular carcinoma caused by HBV is still prevalent globally. We previously reported that interferon (IFN)-induced tripartite motif-containing 25 (TRIM25) inhibited HBV replication by increasing the IFN expression, and this study aimed to further clarify the anti-HBV mechanism of TRIM25.@*METHODS@#The TRIM25-mediated degradation of hepatitis B virus X (HBx) protein was determined by detecting the expression of HBx in TRIM25-overexpressed or knocked-out HepG2 or HepG2-NTCP cells via Western blotting. Co-immunoprecipitation was performed to confirm the interaction between TRIM25 and HBx, and colocalization of TRIM25 and HBx was identified via immunofluorescence; HBV e-antigen and HBV surface antigen were qualified by using an enzyme-linked immunosorbent assay (ELISA) kit from Kehua Biotech. TRIM25 mRNA, pregenomic RNA (pgRNA), and HBV DNA were detected by quantitative real-time polymerase chain reaction. The retinoic acid-inducible gene I (RIG-I) and pgRNA interaction was verified by RNA-binding protein immunoprecipitation assay.@*RESULTS@#We found that TRIM25 promoted HBx degradation, and confirmed that TRIM25 could enhance the K90-site ubiquitination of HBx as well as promote HBx degradation by the proteasome pathway. Interestingly, apart from the Really Interesting New Gene (RING) domain, the SPRY domain of TRIM25 was also indispensable for HBx degradation. In addition, we found that the expression of TRIM25 increased the recognition of HBV pgRNA by interacting with RIG-I, which further increased the IFN production, and SPRY, but not the RING domain is critical in this process.@*CONCLUSIONS@#The study found that TRIM25 interacted with HBx and promoted HBx-K90-site ubiquitination, which led to HBx degradation. On the other hand, TRIM25 may function as an adaptor, which enhanced the recognition of pgRNA by RIG-I, thereby further promoting IFN production. Our study can contribute to a better understanding of host-virus interaction.
Assuntos
Humanos , Vírus da Hepatite B , Proteína DEAD-box 58/metabolismo , RNA , Neoplasias Hepáticas , Replicação Viral , Proteínas com Motivo Tripartido/genética , Fatores de Transcrição , Ubiquitina-Proteína Ligases/genéticaRESUMO
RNA helicases, the largest family of proteins that participate in RNA metabolism, stabilize the intracellular environment through various processes, such as translation and pre-RNA splicing. These proteins are also involved in some diseases, such as cancers and viral diseases. Autophagy, a self-digestive and cytoprotective trafficking process in which superfluous organelles and cellular garbage are degraded to stabilize the internal environment or maintain basic cellular survival, is associated with human diseases. Interestingly, similar to autophagy, RNA helicases play important roles in maintaining cellular homeostasis and are related to many types of diseases. According to recent studies, RNA helicases are closely related to autophagy, participate in regulating autophagy, or serve as a bridge between autophagy and other cellular activities that widely regulate some pathophysiological processes or the development and progression of diseases. Here, we summarize the most recent studies to understand how RNA helicases function as regulatory proteins and determine their association with autophagy in various diseases.
Assuntos
Animais , Humanos , Antivirais/farmacologia , Autofagia , Proteína Beclina-1/metabolismo , Carcinogênese , Sobrevivência Celular , Proteína DEAD-box 58/metabolismo , Progressão da Doença , Regulação da Expressão Gênica , Homeostase , Sistema Imunitário/fisiologia , Neoplasias/metabolismo , RNA Helicases/metabolismo , Splicing de RNA , Receptores Imunológicos/metabolismoRESUMO
Tripartite motif (TRIM) family proteins are important effectors of innate immunity against viral infections. Here we identified TRIM35 as a regulator of TRAF3 activation. Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon (IFN) in response to viral infection. Trim35-deficient mice were more susceptible to influenza A virus (IAV) infection than were wild-type mice. TRIM35 promoted the RIG-I-mediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1. IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3. TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2, thereby antagonizing its suppression of TRAF3 activation. Our in vitro and in vivo findings thus reveal novel roles of TRIM35, through catalyzing Lys63- or Lys48-linked polyubiquitination, in RIG-I antiviral immunity and mechanism of defense against IAV infection.
Assuntos
Animais , Cães , Humanos , Camundongos , Células A549 , Proteínas Reguladoras de Apoptose/imunologia , Proteína DEAD-box 58/imunologia , Células HEK293 , Vírus da Influenza A Subtipo H1N1/imunologia , Células Madin Darby de Rim Canino , Camundongos Knockout , Infecções por Orthomyxoviridae/patologia , Proteólise , Transdução de Sinais/imunologia , Células THP-1 , Fator 3 Associado a Receptor de TNF/imunologia , Ubiquitinação/imunologia , Proteínas Virais/imunologiaRESUMO
It was widely known that retinoic acid inducible gene I (RIG-I) functions as a cytosolic pattern recognition receptor that initiates innate antiviral immunity by detecting exogenous viral RNAs. However, recent studies showed that RIG-I participates in other various cellular activities by sensing endogenous RNAs under different circumstances. For example, RIG-I facilitates the therapy resistance and expansion of breast cancer cells and promotes T cell-independent B cell activation through interferon signaling activation by recognizing non-coding RNAs and endogenous retroviruses in certain situations. While in hepatocellular carcinoma and acute myeloid leukemia, RIG-I acts as a tumor suppressor through either augmenting STAT1 activation by competitively binding STAT1 against its negative regulator SHP1 or inhibiting AKT-mTOR signaling pathway by directly interacting with Src respectively. These new findings suggest that RIG-I plays more diverse roles in various cellular life activities, such as cell proliferation and differentiation, than previously known. Taken together, the function of RIG-I exceeds far beyond that of a pattern recognition receptor.
Assuntos
Animais , Camundongos , Proteína DEAD-box 58 , Genética , Metabolismo , RNA Viral , Genética , Metabolismo , Fator de Transcrição STAT1 , Genética , Metabolismo , Transdução de Sinais , Genética , FisiologiaRESUMO
The innate immune response is the first line of host defense to eliminate viral infection. Pattern recognition receptors in the cytosol, such as RIG-I-like receptors (RLR) and Nod-like receptors (NLR), and membrane bound Toll like receptors (TLR) detect viral infection and initiate transcription of a cohort of antiviral genes, including interferon (IFN) and interferon stimulated genes (ISGs), which ultimately block viral replication. Another mechanism to reduce viral spread is through RIPA, the RLR-induced IRF3-mediated pathway of apoptosis, which causes infected cells to undergo premature death. The transcription factor IRF3 can mediate cellular antiviral responses by both inducing antiviral genes and triggering apoptosis through the activation of RIPA. The mechanism of IRF3 activation in RIPA is distinct from that of transcriptional activation; it requires linear polyubiquitination of specific lysine residues of IRF3. Using RIPA-active, but transcriptionally inactive, IRF3 mutants, it was shown that RIPA can prevent viral replication and pathogenesis in mice.
Assuntos
Animais , Humanos , Camundongos , Apoptose , Proteína DEAD-box 58 , Genética , Alergia e Imunologia , Metabolismo , Imunidade Inata , Fator Regulador 3 de Interferon , Genética , Alergia e Imunologia , Metabolismo , Viroses , Genética , Alergia e Imunologia , MetabolismoRESUMO
Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-β transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.
Assuntos
Animais , Linhagem Celular Tumoral , Clonagem Molecular , Proteína DEAD-box 58 , Genética , Alergia e Imunologia , Fibroblastos , Alergia e Imunologia , Patologia , Expressão Gênica , Hepatite B , Genética , Alergia e Imunologia , Patologia , Vírus da Hepatite B da Marmota , Imunidade Inata , Interferon beta , Genética , Alergia e Imunologia , Ponto Isoelétrico , Rim , Alergia e Imunologia , Patologia , Virologia , Fígado , Alergia e Imunologia , Patologia , Virologia , Marmota , Genética , Alergia e Imunologia , Virologia , Fases de Leitura Aberta , Domínios Proteicos , RNA de Cadeia Dupla , RNA Interferente Pequeno , Genética , Metabolismo , Doenças dos Roedores , Genética , Alergia e Imunologia , Patologia , VirologiaRESUMO
Influenza A virus can create acute respiratory infection in humans and animals throughout the world, and it is still one of the major causes of morbidity and mortality in humans worldwide. Numerous studies have shown that influenza A virus infection induces rapidly host innate immune response. Influenza A virus triggers the activation of signaling pathways that are dependent on host pattern recognition receptors (PRRs) including toll like receptors (TLRs) and RIG-I like receptors (RLRs). Using a variety of regulatory mechanisms, these signaling pathways activate downstream transcript factors that control expression of various interferons and cytokines, such as type I and type III interferons. Thus, these interferons stimulate the transcript of relevant interferon-stimulated genes (ISGs) and expression of the antiviral proteins, which are critical components of host innate immunity. In this review, we will highlight the mechanisms by which influenza A virus infection induces the interferon-mediated host innate immunity.
Assuntos
Humanos , Citocinas , Alergia e Imunologia , Proteína DEAD-box 58 , RNA Helicases DEAD-box , Alergia e Imunologia , Imunidade Inata , Vírus da Influenza A , Influenza Humana , Alergia e Imunologia , Interferons , Alergia e Imunologia , Receptores de Reconhecimento de Padrão , Alergia e Imunologia , Transdução de Sinais , Receptores Toll-Like , Alergia e ImunologiaRESUMO
<p><b>OBJECTIVE</b>To observe the anti-virus effects of andrographolide (AD) on the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) signaling pathway when immunological cells were infected with H1N1.</p><p><b>METHODS</b>Leukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line (16HBE). The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium (MTT) assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunosorbent assay (ELISA) detection while immunological cells for real-time polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-β promoter stimulator-1 (IPS-1), interferon regulatory factor (IRF)-7, IRF-3 and nuclear transcription factor κB (NF-κB) mRNA levels increased significantly (P<0.05). When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF-κB decreased significantly in each group with significant statistic differences (P<0.05).</p><p><b>CONCLUSIONS</b>The RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.</p>
Assuntos
Humanos , Proteínas Adaptadoras de Transdução de Sinal , Genética , Metabolismo , Antivirais , Farmacologia , Células Cultivadas , Técnicas de Cocultura , Proteína DEAD-box 58 , RNA Helicases DEAD-box , Genética , Metabolismo , Células Dendríticas , Alergia e Imunologia , Virologia , Diterpenos , Farmacologia , Sangue Fetal , Biologia Celular , Vírus da Influenza A Subtipo H1N1 , Alergia e Imunologia , Influenza Humana , Tratamento Farmacológico , Alergia e Imunologia , Virologia , Interferon beta , Genética , Metabolismo , Interferon gama , Metabolismo , Interleucina-4 , Metabolismo , Leucócitos Mononucleares , Alergia e Imunologia , Virologia , Macrófagos , Virologia , NF-kappa B , Genética , Metabolismo , Regiões Promotoras Genéticas , Alergia e Imunologia , RNA Mensageiro , Metabolismo , Transdução de Sinais , Genética , Alergia e ImunologiaRESUMO
RIG-I-like receptors (RLRs) belong to pattern recognition receptors, which perform significant roles in antiviral responses. RLRs can initiate a cascade of signaling transduction that induces the production of type I interferon and activates the interferon signaling pathway, ultimately resulting in antiviral responses. In the course of evolution, viruses have been constantly counteracting host immune systems to facilitate their own survival and replication, and have developed a set of antagonistic strategies. These mainly comprise elusion, disguise and attack strategies to eliminate the activation of RLRs. In virus-infected cells, RLRs recognize viral RNA and then induce antiviral responses. A better understanding of viral antagonistic strategies against RLRs will provide insights into the development of new antiviral medicines. This mini-review concludes that there are three main antagonistic strategies by which RNA viruses can counteract the activation of the RLRs pathway. It aims to provide references and insights for similar studies on viral antagonism in an array of RNA viruses.
Assuntos
Humanos , Proteína DEAD-box 58 , RNA Helicases DEAD-box , Genética , Alergia e Imunologia , Interações Hospedeiro-Patógeno , Vírus de RNA , Genética , Alergia e Imunologia , Fisiologia , RNA de Cadeia Dupla , Genética , Alergia e Imunologia , RNA Viral , Genética , Alergia e Imunologia , Viroses , Genética , Alergia e Imunologia , VirologiaRESUMO
Retinoic acid-inducible gene I (RIG-I) is an important pattern recognition receptor that detects viral RNA and triggers the production of type-I interferons through the downstream adaptor MAVS (also called IPS-1, CARDIF, or VISA). A series of structural studies have elaborated some of the mechanisms of dsRNA recognition and activation of RIG-I. Recent studies have proposed that K63-linked ubiquitination of, or unanchored K63-linked polyubiquitin binding to RIG-I positively regulates MAVS-mediated antiviral signaling. Conversely phosphorylation of RIG-I appears to play an inhibitory role in controlling RIG-I antiviral signal transduction. Here we performed a combined structural and biochemical study to further define the regulatory features of RIG-I signaling. ATP and dsRNA binding triggered dimerization of RIG-I with conformational rearrangements of the tandem CARD domains. Full length RIG-I appeared to form a complex with dsRNA in a 2:2 molar ratio. Compared with the previously reported crystal structures of RIG-I in inactive state, our electron microscopic structure of full length RIG-I in complex with blunt-ended dsRNA, for the first time, revealed an exposed active conformation of the CARD domains. Moreover, we found that purified recombinant RIG-I proteins could bind to the CARD domain of MAVS independently of dsRNA, while S8E and T170E phosphorylation-mimicking mutants of RIG-I were defective in binding E3 ligase TRIM25, unanchored K63-linked polyubiquitin, and MAVS regardless of dsRNA. These findings suggested that phosphorylation of RIG inhibited downstream signaling by impairing RIG-I binding with polyubiquitin and its interaction with MAVS.
Assuntos
Humanos , Proteínas Adaptadoras de Transdução de Sinal , Metabolismo , Trifosfato de Adenosina , Metabolismo , Proteína DEAD-box 58 , RNA Helicases DEAD-box , Química , Genética , Metabolismo , Dimerização , Mutagênese Sítio-Dirigida , Fosforilação , Poliubiquitina , Metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA de Cadeia Dupla , Metabolismo , Proteínas Recombinantes , Química , Genética , Transdução de Sinais , Fatores de Transcrição , Metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Metabolismo , UbiquitinaçãoRESUMO
Hepatitis B virus (HBV) infection disrupt the innate immunity response, which may play an important role in the chronic mechanism, while retinoic acid-induced gene I (RIG-I) mediated signaling pathway is one of the most important channel in the innate immunity. HBx and HBV polymerase may disrupt RIG-I mediated signaling pathway. The recent advances about HBV and RIG-I are reviewed in this article.
Assuntos
Humanos , Proteína DEAD-box 58 , RNA Helicases DEAD-box , Metabolismo , Produtos do Gene pol , Metabolismo , Hepatite B , Alergia e Imunologia , Imunidade Inata , Alergia e Imunologia , Transdução de Sinais , Transativadores , MetabolismoRESUMO
Retinoic acid inducible gene-I (RIG-I) is a caspase recruitment domain (CARD) containing protein that acts as an intracellular RNA receptor and senses virus infection. After binding to double stranded RNA (dsRNA) or 5'-triphosphate single stranded RNA (ssRNA), RIG-I transforms into an open conformation, translocates onto mitochondria, and interacts with the downstream adaptor mitochondrial antiviral signaling (MAVS) to induce the production of type I interferon and inflammatory factors via IRF3/7 and NF-κB pathways, respectively. Recently, accumulating evidence suggests that RIG-I could function in non-viral systems and participate in a series of biological events, such as inflammation and inflammation related diseases, cell proliferation, apoptosis and even senescence. Here we review recent advances in antiviral study of RIG-I as well as the functions of RIG-I in other fields.
Assuntos
Humanos , Antivirais , Química , Proteína DEAD-box 58 , RNA Helicases DEAD-box , Química , Metabolismo , Fisiologia , Inflamação , Metabolismo , Fator Regulador 3 de Interferon , Metabolismo , NF-kappa B , Metabolismo , Vírus de RNA , Metabolismo , RNA de Cadeia Dupla , Metabolismo , Transdução de SinaisRESUMO
Hepatitis B virus (HBV) is regarded as a stealth virus, invading and replicating efficiently in human liver undetected by host innate antiviral immunity. Here, we show that type I interferon (IFN) induction but not its downstream signaling is blocked by HBV replication in HepG2.2.15 cells. This effect may be partially due to HBV X protein (HBx), which impairs IFNβ promoter activation by both Sendai virus (SeV) and components implicated in signaling by viral sensors. As a deubiquitinating enzyme (DUB), HBx cleaves Lys63-linked polyubiquitin chains from many proteins except TANK-binding kinase 1 (TBK1). It binds and deconjugates retinoic acid-inducible gene I (RIG I) and TNF receptor-associated factor 3 (TRAF3), causing their dissociation from the downstream adaptor CARDIF or TBK1 kinase. In addition to RIG I and TRAF3, HBx also interacts with CARDIF, TRIF, NEMO, TBK1, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKi) and interferon regulatory factor 3 (IRF3). Our data indicate that multiple points of signaling pathways can be targeted by HBx to negatively regulate production of type I IFN.
Assuntos
Animais , Humanos , Camundongos , Linfócitos B , Alergia e Imunologia , Metabolismo , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box , Alergia e Imunologia , Metabolismo , Células Hep G2 , Vírus da Hepatite B , Alergia e Imunologia , Metabolismo , Quinase I-kappa B , Alergia e Imunologia , Metabolismo , Evasão da Resposta Imune , Imunidade Inata , Fator Regulador 3 de Interferon , Alergia e Imunologia , Metabolismo , Interferon Tipo I , Alergia e Imunologia , Metabolismo , Terapia de Alvo Molecular , Poliubiquitina , Metabolismo , Ligação Proteica , Alergia e Imunologia , Vírus Sendai , Alergia e Imunologia , Metabolismo , Transdução de Sinais , Alergia e Imunologia , Fator 3 Associado a Receptor de TNF , Alergia e Imunologia , Metabolismo , Transativadores , Alergia e Imunologia , MetabolismoRESUMO
Retinoic acid-inducible gene-I (RIG-I) functions as an intracellular pattern recognition receptor (PRR) that recognizes the 5'-triphosphate moiety of single-stranded RNA viruses to initiate the innate immune response. Previous studies have shown that Lys63-linked ubiquitylation is required for RIG-I activation and the downstream anti-viral type I interferon (IFN-I) induction. Herein we reported that, RIG-I was also modified by small ubiquitin-like modifier-1 (SUMO-1). Functional analysis showed that RIG-I SUMOylation enhanced IFN-I production through increased ubiquitylation and the interaction with its downstream adaptor molecule Cardif. Our results therefore suggested that SUMOylation might serve as an additional regulatory tier for RIG-I activation and IFN-I signaling.