RESUMEN
RNA helicases are involved in the innate immune response against pathogens, including bacteria and viruses; however, their mechanism in the human airway epithelial cells is still not fully understood. Here, we demonstrated that DEAH (Asp-Glu-Ala-His) box polypeptide 35 (DHX35), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family, boosts antiviral innate immunity in human airway epithelial cells. DHX35 knockdown attenuated the production of interferon-ß (IFN-ß), IL6, and CXCL10, whereas DHX35 overexpression increased their production. Upon stimulation, DHX35 was constitutively expressed, but it translocated from the nucleus into the cytosol, where it recognized cytosolic poly(I:C) and poly(dA:dT) via its HELICc domain. Mitochondrial antiviral signaling protein (MAVS) acted as an adaptor for DHX35 and interacted with the HELICc domain of DHX35 using amino acids 360-510. Interestingly, DHX35 interacted with retinoic acid-inducible gene 1 (RIG-I), enhanced the binding affinity of RIG-I with poly(I:C) and poly(dA:dT), and formed a signalsome with MAVS to activate interferon regulatory factor 3 (IRF3), NF-κB-p65, and MAPK signaling pathways. These results indicate that DHX35 not only acted as a cytosolic nucleic acid sensor but also synergized with RIG-I to enhance antiviral immunity in human airway epithelial cells. Our results demonstrate a novel molecular mechanism for DHX35 in RIG-I-mediated innate immunity and provide a novel candidate for drug and vaccine design to control viral infections in the human airway.
Asunto(s)
Proteína 58 DEAD Box , ARN Helicasas DEAD-box , Inmunidad Innata , Receptores Inmunológicos , Humanos , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/inmunología , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/inmunología , Receptores Inmunológicos/metabolismo , Poli I-C/inmunología , Poli I-C/farmacología , ARN Helicasas/metabolismo , ARN Helicasas/inmunología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/inmunología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Células HEK293RESUMEN
RLR helicases RIG-I and MDA5, which are known as pattern recognition receptors to sense cytoplasmic viral RNAs and trigger antiviral immune responses, are DExD/H-box helicases. In teleost, whether and how non-RLR helicases regulate RLR helicases to affect viral infection remains unclear. Here, we report that the non-RLR helicase DHX40 from grass carp (namely gcDHX40) is a negative regulator of grass carp reovirus (GCRV) infection and RLR-mediated type I IFN production. GcDHX40 was a cytoplasmic protein. Ectopic expression of gcDHX40 facilitated GCRV replication and suppressed type I IFN production induced by GCRV infection and by those genes involved the RLR antiviral signaling pathway. Mechanistically, gcDHX40 promoted the generation of viral inclusion bodies (VIBs) by interacting with the NS38 protein of GCRV. Additionally, gcDHX40 interacted with RLR helicase, and impaired the formation of RLR-MAVS functional complexes. Taken together, our results indicate that gcDHX40 is a novel important proviral host factor involving in promoting the generation of GCRV VIBs and inhibiting the production of RLR-mediated type I IFNs.
Asunto(s)
Carpas , ARN Helicasas DEAD-box , Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Infecciones por Reoviridae , Reoviridae , Proteínas no Estructurales Virales , Animales , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismo , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Carpas/inmunología , Carpas/genética , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/inmunología , Reoviridae/fisiología , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/inmunología , ARN Helicasas DEAD-box/metabolismo , Inmunidad Innata/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Helicasas/inmunología , Regulación de la Expresión Génica/inmunologíaRESUMEN
Type I interferons (IFN-I) are essential to establish antiviral innate immunity. Unanchored (or free) polyubiquitin (poly-Ub) has been shown to regulate IFN-I responses. However, few unanchored poly-Ub interactors are known. To identify factors regulated by unanchored poly-Ub in a physiological setting, we developed an approach to isolate unanchored poly-Ub from lung tissue. We identified the RNA helicase DHX16 as a potential pattern recognition receptor (PRR). Silencing of DHX16 in cells and in vivo diminished IFN-I responses against influenza virus. These effects extended to members of other virus families, including Zika and SARS-CoV-2. DHX16-dependent IFN-I production requires RIG-I and unanchored K48-poly-Ub synthesized by the E3-Ub ligase TRIM6. DHX16 recognizes a signal in influenza RNA segments that undergo splicing and requires its RNA helicase motif for direct, high-affinity interactions with specific viral RNAs. Our study establishes DHX16 as a PRR that partners with RIG-I for optimal activation of antiviral immunity requiring unanchored poly-Ub.
Asunto(s)
Proteína 58 DEAD Box , Interferón Tipo I , ARN Helicasas , ARN Viral , Receptores Inmunológicos , Infección por el Virus Zika , Virus Zika , COVID-19 , Proteína 58 DEAD Box/inmunología , Humanos , Inmunidad Innata , Interferón Tipo I/inmunología , ARN Helicasas/inmunología , Receptores Inmunológicos/inmunología , SARS-CoV-2 , Proteínas de Motivos Tripartitos , Virus Zika/genética , Infección por el Virus Zika/inmunologíaRESUMEN
Natural killer (NK) cells are innate immune cells that contribute to host defense against virus infections. NK cells respond to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and are activated in patients with acute coronavirus disease 2019 (COVID-19). However, by which mechanisms NK cells detect SARS-CoV-2-infected cells remains largely unknown. Here, we show that the Non-structural protein 13 of SARS-CoV-2 encodes for a peptide that is presented by human leukocyte antigen E (HLA-E). In contrast with self-peptides, the viral peptide prevents binding of HLA-E to the inhibitory receptor NKG2A, thereby rendering target cells susceptible to NK cell attack. In line with these observations, NKG2A-expressing NK cells are particularly activated in patients with COVID-19 and proficiently limit SARS-CoV-2 replication in infected lung epithelial cells in vitro. Thus, these data suggest that a viral peptide presented by HLA-E abrogates inhibition of NKG2A+ NK cells, resulting in missing self-recognition.
Asunto(s)
COVID-19 , Antígenos de Histocompatibilidad Clase I , Células Asesinas Naturales , Metiltransferasas , Subfamília C de Receptores Similares a Lectina de Células NK , ARN Helicasas , SARS-CoV-2 , Proteínas no Estructurales Virales , COVID-19/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Células Asesinas Naturales/inmunología , Metiltransferasas/inmunología , Subfamília C de Receptores Similares a Lectina de Células NK/inmunología , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismo , Péptidos/metabolismo , ARN Helicasas/inmunología , Proteínas no Estructurales Virales/inmunología , Antígenos HLA-ERESUMEN
As a highly pathogenic human coronavirus, SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread. The nucleic acid-induced stress response is an essential component of antiviral defense and is closely related to antiviral innate immunity. However, whether SARS-CoV-2 regulates the stress response pathway to achieve immune evasion remains elusive. In this study, SARS-CoV-2 NSP5 and N protein were found to attenuate antiviral stress granule (avSG) formation. Moreover, NSP5 and N suppressed IFN expression induced by infection of Sendai virus or transfection of a synthetic mimic of dsRNA, poly (I:C), inhibiting TBK1 and IRF3 phosphorylation, and restraining the nuclear translocalization of IRF3. Furthermore, HEK293T cells with ectopic expression of NSP5 or N protein were less resistant to vesicular stomatitis virus infection. Mechanistically, NSP5 suppressed avSG formation and disrupted RIG-I-MAVS complex to attenuate the RIG-I-mediated antiviral immunity. In contrast to the multiple targets of NSP5, the N protein specifically targeted cofactors upstream of RIG-I. The N protein interacted with G3BP1 to prevent avSG formation and to keep the cofactors G3BP1 and PACT from activating RIG-I. Additionally, the N protein also affected the recognition of dsRNA by RIG-I. This study revealed the intimate correlation between SARS-CoV-2, the stress response, and innate antiviral immunity, shedding light on the pathogenic mechanism of COVID-19.
Asunto(s)
Proteasas 3C de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/genética , Proteína 58 DEAD Box/genética , ADN Helicasas/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas de Unión al ARN/genética , Receptores Inmunológicos/genética , SARS-CoV-2/genética , Gránulos de Estrés/genética , Animales , Chlorocebus aethiops , Proteasas 3C de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteína 58 DEAD Box/inmunología , ADN Helicasas/inmunología , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Evasión Inmune , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Poli I-C/farmacología , Proteínas de Unión a Poli-ADP-Ribosa/inmunología , Unión Proteica , ARN Helicasas/inmunología , Proteínas con Motivos de Reconocimiento de ARN/inmunología , ARN Bicatenario/genética , ARN Bicatenario/inmunología , Proteínas de Unión al ARN/inmunología , Receptores Inmunológicos/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Virus Sendai/genética , Virus Sendai/inmunología , Transducción de Señal , Gránulos de Estrés/efectos de los fármacos , Gránulos de Estrés/inmunología , Gránulos de Estrés/virología , Células Vero , Vesiculovirus/genética , Vesiculovirus/inmunologíaRESUMEN
Stress granules (SGs) are highly dynamic cytoplasmic foci that form in response to activation of the integrated stress response (ISR) that results in eIF2α phosphorylation and global translation shutdown. Stress granules, which are largely nucleated by G3BP1, serve as hubs for mRNA triage, but there is mounting evidence that they also perform cell signaling functions that are vital to cell survival, particularly during viral infection. We previously showed that SG formation leads to NFκB activation and JNK signaling and that this association may be due in part to G3BP1-dependent recruitment of PKR to SGs. Others have reported close associations between G3BP1 and various innate immune PRRs of the type 1 interferon signaling system, including RIG-I. We also reported SG assembly dynamics is dependent on the arginine-methylation status of G3BP1. Another protein that rapidly localizes to SGs, TDRD3, is a methyl reader protein that performs transcriptional activation and adaptor functions within the nucleus, but neither the mechanism nor its function in SGs is clear. Here, we present evidence that TDRD3 localizes to SGs partly based upon methylation potential of G3BP1. We also characterize granules that TDRD3 forms during overexpression and show that these granules can form in the absence of G3BP but also contain translation components found in canonical SGs. We also show for the first time that SGs recruit additional interferon effectors IRF3, IRF7, TBK1, and Sting, and provide evidence that TDRD3 may play a role in recruitment of these factors. We also present evidence that TDRD3 is a novel antiviral protein that is cleaved by enteroviral 2A proteinase. G3BP1 and TDRD3 knockdown in cells results in altered transcriptional regulation of numerous IFN effectors in complex modulatory patterns that are distinctive for G3BP1 and TDRD3. Overall, we describe a novel role of TDRD3 in innate immunity in which G3BP1 and TDRD3 may coordinate to play important roles in regulation of innate antiviral defenses.
Asunto(s)
ADN Helicasas/inmunología , Inmunidad Innata/inmunología , Proteínas de Unión a Poli-ADP-Ribosa/inmunología , Proteínas/inmunología , ARN Helicasas/inmunología , Proteínas con Motivos de Reconocimiento de ARN/inmunología , Virosis/inmunología , Línea Celular , Humanos , Interferones/inmunología , Transducción de Señal/inmunología , Gránulos de Estrés/inmunologíaRESUMEN
One of the first layers of protection that metazoans put in place to defend themselves against viruses rely on the use of proteins containing DExD/H-box helicase domains. These members of the duplex RNA-activated ATPase (DRA) family act as sensors of double-stranded RNA (dsRNA) molecules, a universal marker of viral infections. DRAs can be classified into 2 subgroups based on their mode of action: They can either act directly on the dsRNA, or they can trigger a signaling cascade. In the first group, the type III ribonuclease Dicer plays a key role to activate the antiviral RNA interference (RNAi) pathway by cleaving the viral dsRNA into small interfering RNAs (siRNAs). This represents the main innate antiviral immune mechanism in arthropods and nematodes. Even though Dicer is present and functional in mammals, the second group of DRAs, containing the RIG-I-like RNA helicases, appears to have functionally replaced RNAi and activate type I interferon (IFN) response upon dsRNA sensing. However, recent findings tend to blur the frontier between these 2 mechanisms, thereby highlighting the crucial and diverse roles played by RNA helicases in antiviral innate immunity. Here, we will review our current knowledge of the importance of these key proteins in viral infection, with a special focus on the interplay between the 2 main types of response that are activated by dsRNA.
Asunto(s)
Inmunidad Innata/inmunología , ARN Helicasas/inmunología , Virosis/inmunología , Animales , HumanosRESUMEN
RNA helicases play roles in various essential biological processes such as RNA splicing and editing. Recent in vitro studies show that RNA helicases are involved in immune responses toward viruses, serving as viral RNA sensors or immune signaling adaptors. However, there is still a lack of in vivo data to support the tissue- or cell-specific function of RNA helicases owing to the lethality of mice with complete knockout of RNA helicases; further, there is a lack of evidence about the antibacterial role of helicases. Here, we investigated the in vivo role of Dhx15 in intestinal antibacterial responses by generating mice that were intestinal epithelial cell (IEC)-specific deficient for Dhx15 (Dhx15 f/f Villin1-cre, Dhx15ΔIEC). These mice are susceptible to infection with enteric bacteria Citrobacter rodentium (C. rod), owing to impaired α-defensin production by Paneth cells. Moreover, mice with Paneth cell-specific depletion of Dhx15 (Dhx15 f/f Defensinα6-cre, Dhx15ΔPaneth) are more susceptible to DSS (dextran sodium sulfate)-induced colitis, which phenocopy Dhx15ΔIEC mice, due to the dysbiosis of the intestinal microbiota. In humans, reduced protein levels of Dhx15 are found in ulcerative colitis (UC) patients. Taken together, our findings identify a key regulator of Wnt-induced α-defensins in Paneth cells and offer insights into its role in the antimicrobial response as well as intestinal inflammation.
Asunto(s)
Colitis/inmunología , Defensinas/genética , Infecciones por Enterobacteriaceae/inmunología , Células de Paneth/inmunología , ARN Helicasas/genética , Vía de Señalización Wnt , Animales , Citrobacter rodentium/inmunología , Citrobacter rodentium/patogenicidad , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Defensinas/inmunología , Sulfato de Dextran/administración & dosificación , Infecciones por Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/patología , Microbioma Gastrointestinal/inmunología , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/inmunología , Células de Paneth/microbiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , ARN Helicasas/inmunologíaRESUMEN
Dengue virus reportedly circulates as four genetically distinct serotypes for which there is no widely accepted vaccine or drug at present. Morbidity and mortality caused by this virus are alarming for the possible increased threat to human health. A suitable diagnostic test is the prerequisite for designing and developing control measures. But, the tests being employed at present possess one or the other drawback for this disease diagnosis. During the dengue virus infections, NS2B is essential for the stability and catalytic activity of the NS3 protease. N-terminal 185 amino acids of NS3 protease domain along with hydrophilic portion of NS2B (NS2BNS3pro) is being used to screen dengue inhibitors but not for diagnosis until now. In the present study, we have used purified NS2BNS3pro as an antigen to trap anti-NS2BNS3pro antibodies of the clinical samples. Antibodies were detected successfully in both Western blot analysis and enzyme-linked immunosorbent assay (ELISA) tests. In ELISA, antibodies were detected in both primary and secondary infections of all serotypes. Interestingly, 17 samples declared as other febrile infections by NS1 and IgM/IgG tests were found to be positive in present test, which were further confirmed by reverse-transcription polymerase chain reaction. In silico studies suggested the absence of conserved epitopes between NS2BNS3pro and the counterpart in JEV, Zika, and CHIKV, indicating less possibility of crossreaction, which was in turn confirmed by using synthetic peptides representing the above epitopes. Statistical analysis with 76% specificity, 87% sensitivity, and 95% concordance also supported the present test as a suitable test for large scale diagnosis of dengue virus infections.
Asunto(s)
Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Dengue/diagnóstico , Dengue/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas no Estructurales Virales/inmunología , Simulación por Computador , Virus del Dengue/química , Virus del Dengue/genética , Epítopos/química , Epítopos/inmunología , Humanos , Inmunoglobulina M/inmunología , ARN Helicasas/inmunología , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad , Serina Endopeptidasas/inmunologíaRESUMEN
Porcine epidemic diarrhea virus (PEDV) is an enteric swine coronavirus. Recent PEDV eruption in East Asian and North American countries made it notorious and caused huge economic losses to the porcine industry. Nonstructural protein 13 (nsp13) is a nucleic acid helicase/NTPase that plays a critical role in viral gene transcription and viral replication. To investigate the function of nsp13 in the context of PEDV infection, here, PEDV nsp13 gene was amplified and cloned into pET28a/pET30a/pGEX-6P-1 expression vectors. The recombinant his-tagged nsp13 and GST-tagged nsp13 were expressed. Purified his-tagged nsp13 from pET28a-nsp13 vectors was chosen for immunization. Three monoclonal antibodies (mAbs) named 5A9, 5C7, and 5G7 were identified from the hybridoma cells, and were characterized by Western blot analysis and immunofluorescent assay, which demonstrated high specificity of these three mAbs with pCAGGS-HA-nsp13. All three mAbs belong to IgG1+ kappa subclass. However, only mAb 5A9 could effectively and specifically recognize PEDV expressing nsp13. Furthermore, the generated antibody against nsp13 could be applied to investigate nsp13 function during PEDV replication.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Gastroenteritis Porcina Transmisible/terapia , Virus de la Diarrea Epidémica Porcina/inmunología , Proteínas no Estructurales Virales/inmunología , Animales , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/uso terapéutico , Línea Celular , Femenino , Células HEK293 , Humanos , Inmunización , Metiltransferasas/inmunología , Ratones , Ratones Endogámicos BALB C , ARN Helicasas/inmunología , PorcinosRESUMEN
No disponible
Asunto(s)
Humanos , ARN Viral/análisis , Infecciones por Coronavirus/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Neumonía Viral/inmunología , Autoinmunidad , ARN Helicasas/inmunología , Receptores Toll-Like/inmunología , Interacciones Huésped-Patógeno/efectos de los fármacos , Antivirales/farmacocinética , Infecciones por Coronavirus/tratamiento farmacológico , Corticoesteroides/uso terapéutico , Hidroxicloroquina/uso terapéutico , Sulfasalazina/uso terapéutico , Metotrexato/uso terapéutico , Ciclosporina/uso terapéutico , Colchicina/uso terapéutico , Azitromicina/uso terapéuticoRESUMEN
RIG-I, MDA5, and LGP2 comprise the RIG-I-like receptors (RLRs). RIG-I and MDA5 are essential pathogen recognition receptors sensing viral infections while LGP2 has been described as both RLR cofactor and negative regulator. After sensing and binding to viral RNA, including double-stranded RNA (dsRNA), RIG-I and MDA5 undergo cytosol-to-membrane relocalization to bind and signal through the MAVS adaptor protein on intracellular membranes, thus directing downstream activation of IRF3 and innate immunity. Here, we report examination of the dynamic subcellular localization of all three RLRs within the intracellular response to dsRNA and RNA virus infection. Observations from high resolution biochemical fractionation and electron microscopy, coupled with analysis of protein interactions and IRF3 activation, show that, in resting cells, microsome but not mitochondrial fractions harbor the central components to initiate innate immune signaling. LGP2 interacts with MAVS in microsomes, blocking the RIG-I/MAVS interaction. Remarkably, in response to dsRNA treatment or RNA virus infection, LGP2 is rapidly released from MAVS and redistributed to mitochondria, temporally correlating with IRF3 activation. We reveal that IRF3 activation does not take place on mitochondria but instead occurs at endoplasmic reticulum (ER)-derived membranes. Our observations suggest ER-derived membranes as key RLR signaling platforms controlled through inhibitory actions of LGP2 binding to MAVS wherein LGP2 translocation to mitochondria releases MAVS inhibition to facilitate RLR-mediated signaling of innate immunity.
Asunto(s)
Proteína 58 DEAD Box/genética , Helicasa Inducida por Interferón IFIH1/genética , ARN Helicasas/genética , Virosis/inmunología , Proteína 58 DEAD Box/inmunología , Humanos , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Factor 3 Regulador del Interferón/genética , Helicasa Inducida por Interferón IFIH1/inmunología , Mitocondrias/genética , Mitocondrias/inmunología , ARN Helicasas/inmunología , ARN Bicatenario/genética , ARN Viral/genética , ARN Viral/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Virosis/genética , Virosis/virologíaRESUMEN
Multifunctional interleukin 10 (IL10)+ Th1 cells have been implicated in favorable evolution of many infectious diseases, promoting an efficacious immune response while limiting immunopathology. Here, we investigated the presence of multifunctional CD4+ and CD8+ T-cells that expressed interferon gamma (IFNγ), IL10 and tumor necrosis factor (TNF), or its combinations during dengue infection. Peripheral blood mononuclear cells (PBMCs) from outpatients with dengue (mild dengue forms) and hospitalized patients (or patients with dengue with warning signs and severe dengue) were cultured in the presence of envelope (ENV) or NS3 peptide libraries of DENV during critical (hospitalization period) and convalescence phases. The production of IFNγ, IL10 and TNF by CD4+ and CD8+ T-cells was assessed by flow cytometry. Our data show that patients with mild dengue, when compared with patients with dengue with warning signs and severe dengue, presented higher frequencies of multifunctional T-cells like NS3-specific IFNγ/IL10-producing CD4+ T-cells in critical phase and NS3- and ENV-specific CD8+ T-cells producing IFNγ/IL10. In addition, NS3-specific CD8+ T-cells producing high levels of IFNγ/TNF and IFNγ/TNF/IL10 were also observed in the mild dengue group. We observed that multifunctional T-cells produced higher levels of cytokines as measured by intracellular content when compared with single producer T-cells. Importantly, multifunctional CD4+ and CD8+ T-cells producing IFNγ, TNF and IL10 simultaneously displayed positive correlation with platelet levels, suggesting a protective role of this population. The presence of IL10+ Th1 and IL10+ Tc1 multifunctional cells was associated with mild dengue presentation, suggesting that these cells play a role in clinical evolution of dengue infection.
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Dengue/diagnóstico , Dengue/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Anciano , Antígenos Virales/inmunología , Brasil , Estudios de Casos y Controles , Dengue/sangre , Virus del Dengue/inmunología , Femenino , Voluntarios Sanos , Humanos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Masculino , Persona de Mediana Edad , Cultivo Primario de Células , ARN Helicasas/inmunología , Serina Endopeptidasas/inmunología , Índice de Severidad de la Enfermedad , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas no Estructurales Virales/inmunología , Adulto JovenRESUMEN
Japanese encephalitis (JE) is a serious leading health complication emerging expansively that has severely affected the survival rate of human beings. This fatal disease is caused by JE Virus (JEV). The current study was carried out for designing a multi-epitope loaded peptide vaccine to prevent JEV. Based on reverse vaccinology and in silico approaches, octapeptide B-cell and hexapeptide T-cell epitopes belonging to five proteins, viz. E, prM, NS1, NS3 and NS5 of JEV were determined. Hydrophilicity, antigenicity, immunogenicity and aliphatic amino acids of the epitopes were estimated. Further, the epitopes were analyzed for different physicochemical parameters, e.g. total net charges, amino acid composition and Boman index. Out of all the epitopes, a total of four T-cell epitopes namely KRADSS, KRSRRS, SKRSRR and KECPDE and one B-cell epitope i.e. PKPCSKGD were found to have potential for raising immunity in human against the pathogen. Taking into account the outcome of this study, the pharmaceutical industries could initiate efforts to combine the identified epitopes together with adjuvant or carrier protein to develop a multi-epitope-loaded peptide vaccine against JEV. The peptide vaccine, being cost effective, could be administered as a prophylactic measure and in JEV infected individuals to combat the spread of this virus in human population. However, prior to administration into human beings, the vaccine must pass through several clinical trials.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie)/inmunología , Vacunas contra la Encefalitis Japonesa/inmunología , Aminoácidos/análisis , Linfocitos B/inmunología , Epítopos/química , Epítopos/inmunología , Inmunogenicidad Vacunal , Péptidos/inmunología , ARN Helicasas/inmunología , Serina Endopeptidasas/inmunología , Proteínas no Estructurales Virales/inmunologíaRESUMEN
INTRODUCTION: Although the role of dengue virus (DENV)-specific T cells in the pathogenesis of acute dengue infection is emerging, the functionality of virus-specific T cells associated with milder clinical disease has not been well studied. We sought to investigate how the functionality of DENV-NS3 and DENV-NS5 protein-specific T cells differ in patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). METHODS: Using intracellular cytokine assays, we assessed the production of interferon γ (IFNγ), tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-1ß (MIP-1ß), and CD107a expression in adult patients with acute DF (n = 21) and DHF (n = 22). RESULTS: Quadruple cytokine-producing, polyfunctional DENV-NS3- and DENV-NS5-specific T cells were more frequent in those with DF when compared to those with DHF. While DENV-NS3- and DENV-NS5-specific T cells in patients with DF expressed IFNγ > TNF-α > MIP-ß > CD107a, T cells of those with DHF predominantly expressed CD107a > MIP-1ß > IFNγ > TNF-α. Overall production of IFNγ or TNF-α by DENV-NS3- and DENV-NS5-specific T cells was significantly higher in patients with DF. The majority of NS3-specific T cells in patients with DF (78.6%) and DHF (68.9%) were single-cytokine producers; 76.6% of DENV-NS5-specific T cells in those with DF and 77.1% of those with DHF, produced only a single cytokine. However, no significant association was found with polyfunctional T-cell responses and the degree of viraemia. CONCLUSIONS: Our results suggest that the functional phenotype of DENV-specific T cells are likely to associate with clinical disease severity.
Asunto(s)
Citocinas/inmunología , Dengue/inmunología , Inmunidad Celular , Linfocitos T/inmunología , Proteínas no Estructurales Virales/inmunología , Enfermedad Aguda , Adulto , Dengue/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , ARN Helicasas/inmunología , Serina Endopeptidasas/inmunología , Índice de Severidad de la Enfermedad , Linfocitos T/patologíaRESUMEN
The emergence of novel and divergent HoBi-like pestivirus (HoBiPeV) strains in cattle in Asia recently has raised concerns with regard to their reliable and accurate diagnosis. Hence, the aim of this study was to evaluate currently available BVDV diagnostic tests and HoBiPeV-specific diagnostic tests in detection of genetically divergent strains of HoBiPeV. One strain each of HoBiPeV-c and d were subjected to two BVDV diagnostic RT-PCR tests, one HoBiPeV specific RT-PCR test, three BVDV diagnostic qRT-PCR tests, one HoBiPeV specific qRT-PCR test and two BVDV antigen capture ELISAs. Archived cattle sera (nâ¯=â¯41) from farms with reports of HoBiPeV natural infection were assessed for detection of HoBiPeV antibodies by VNT and two commercial BVD antibody ELISA kits. BVDV diagnostic qRT-PCR tests had better sensitivity than BVDV diagnostic RT-PCR tests, while majority of them except a commercial kit showed a lower sensitivity for HoBiPeV-d strain. The HoBiPeV specific qRT-PCR test was found more sensitive than HoBiPeV specific RT-PCR but both had lower sensitivity for HoBiPeV-d strain, as displayed by primer/probe sequence mismatches. The BVDV Erns antigen ELISA detected both the strains of HoBiPeV, but with a lower sensitivity for HoBiPeV-d strain, whereas BVDV NS3 antigen ELISA failed to detect them even at a high HoBiPeV titre. Compared to VNT, commercial BVDV antibody ELISA showed low to moderate sensitivity in detection of HoBiPeV antibodies, with a failure rate of 31.25% for the whole virus antigen based ELISA and a failure rate of 56.25% for NS3 antibody ELISA. The present study demonstrated new challenges in HoBiPeV diagnosis indicating a need in improvement of both HoBiPeV specific diagnostic RT-PCR and qRT-PCR for better utility in HoBiPeV epidemiology and biological product safety. Although more studies are required, this study reinforces that combined use of BVDV Erns and NS3 antigen ELISA may have some utility in preliminary differentiation between HoBiPeV and BVDV infection in PI cattle. Additionally, we show that the comparative VNT has a better sensitivity in detection of HoBiPeV exposure and there is a need of robust antibody ELISA for reliable detection of antibodies against this emerging bovine pestivirus.
Asunto(s)
Diarrea Mucosa Bovina Viral/diagnóstico , Pruebas Diagnósticas de Rutina/métodos , Pruebas Diagnósticas de Rutina/veterinaria , Virus de la Diarrea Viral Bovina/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Pestivirus/aislamiento & purificación , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/sangre , Antígenos Virales/inmunología , Diarrea Mucosa Bovina Viral/inmunología , Diarrea Mucosa Bovina Viral/virología , Bovinos , Virus de la Diarrea Viral Bovina/inmunología , Pruebas de Neutralización , Péptido Hidrolasas/inmunología , Pestivirus/inmunología , ARN Helicasas/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas no Estructurales Virales/inmunologíaRESUMEN
The integrated stress response (ISR) is a cellular response system activated upon different types of stresses, including viral infection, to restore cellular homeostasis. However, many viruses manipulate this response for their own advantage. In this study, we investigated the association between murine norovirus (MNV) infection and the ISR and demonstrate that MNV regulates the ISR by activating and recruiting key ISR host factors. We observed that during MNV infection, there is a progressive increase in phosphorylated eukaryotic initiation factor 2α (p-eIF2α), resulting in the suppression of host translation, and yet MNV translation still progresses under these conditions. Interestingly, the shutoff of host translation also impacts the translation of key signaling cytokines such as beta interferon, interleukin-6, and tumor necrosis factor alpha. Our subsequent analyses revealed that the phosphorylation of eIF2α was mediated via protein kinase R (PKR), but further investigation revealed that PKR activation, phosphorylation of eIF2α, and translational arrest were uncoupled during infection. We further observed that stress granules (SGs) are not induced during MNV infection and that MNV can restrict SG nucleation and formation. We observed that MNV recruited the key SG nucleating protein G3BP1 to its replication sites and intriguingly the silencing of G3BP1 negatively impacts MNV replication. Thus, it appears that MNV utilizes G3BP1 to enhance replication but equally to prevent SG formation, suggesting an anti-MNV property of SGs. Overall, this study highlights MNV manipulation of SGs, PKR, and translational control to regulate cytokine translation and to promote viral replication.IMPORTANCE Viruses hijack host machinery and regulate cellular homeostasis to actively replicate their genome, propagate, and cause disease. In retaliation, cells possess various defense mechanisms to detect, destroy, and clear infecting viruses, as well as signal to neighboring cells to inform them of the imminent threat. In this study, we demonstrate that the murine norovirus (MNV) infection stalls host protein translation and the production of antiviral and proinflammatory cytokines. However, virus replication and protein translation still ensue. We show that MNV further prevents the formation of cytoplasmic RNA granules, called stress granules (SGs), by recruiting the key host protein G3BP1 to the MNV replication complex, a recruitment that is crucial to establishing and maintaining virus replication. Thus, MNV promotes immune evasion of the virus by altering protein translation. Together, this evasion strategy delays innate immune responses to MNV infection and accelerates disease onset.
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Infecciones por Caliciviridae/inmunología , Gránulos Citoplasmáticos/virología , ADN Helicasas/inmunología , Factor 2 Eucariótico de Iniciación/inmunología , Evasión Inmune , Proteínas de Unión a Poli-ADP-Ribosa/inmunología , ARN Helicasas/inmunología , Proteínas con Motivos de Reconocimiento de ARN/inmunología , eIF-2 Quinasa/inmunología , Animales , Gránulos Citoplasmáticos/inmunología , Interacciones Huésped-Patógeno , Inmunidad Innata , Ratones , Fosforilación , Biosíntesis de Proteínas , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
RNA helicases play an important role in the response to microbial infection. Retinoic acid inducible gene-I (RIG-I) and members of the RIG-I-like receptor (RLR) family of helicases function as cytoplasmic pattern recognition receptors (PRRs) whose actions are essential for recognition of RNA viruses. RIG-I association with pathogen-associated molecular patterns (PAMPs) within viral RNA leads to its activation and signaling via the mitochondrial antiviral signaling (MAVS) adapter protein. This interaction mediates downstream signaling events that drive the innate immune response to virus infection. Here we identify the DEAH-box RNA helicase DHX15 as a RLR binding partner and signaling cofactor. In human cells, DHX15 is required for virus-induced RLR signaling of innate immune gene expression. Knockdown of DHX15 increased susceptibility to infection by RNA viruses of diverse genera, including Paramyxoviridae, Rhabdoviridae, and Picornaviridae. DHX15 associates with RIG-I caspase activation and recruitment domains (CARDs) through its amino terminus, in which the complex is recruited to MAVS on virus infection. Importantly, although DHX15 cannot substitute for RIG-I in innate immune signaling, DHX15 selectively binds PAMP RNA to promote RIG-I ATP hydrolysis and signaling activation in response to viral RNA. Our results define DHX15 as a coreceptor required for RLR innate immune responses to control RNA virus infection.
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ARN Helicasas/inmunología , Infecciones por Virus ARN/inmunología , Receptores de Reconocimiento de Patrones/inmunología , Virus Sendai/inmunología , Transducción de Señal/inmunología , Células Cultivadas , Células HEK293 , HumanosRESUMEN
The stress response genes encoding GADD45γ, and to a lesser extent GADD45ß, are activated early in infection with herpes simplex virus 1 (HSV-1). Cells that had been depleted of GADD45γ by transfection of short hairpin RNA (shRNA) or in which the gene had been knocked out (ΔGADD45γ) yielded significantly less virus than untreated infected cells. Consistent with lower virus yields, the ΔGADD45γ cells (either uninfected or infected with HSV-1) exhibited significantly higher levels of transcripts of a cluster of innate immunity genes, including those encoding IFI16, IFIT1, MDA5, and RIG-I. Members of this cluster of genes were reported by this laboratory to be activated concurrently with significantly reduced virus yields in cells depleted of LGP2 or HDAC4. We conclude that innate immunity to HSV-1 is normally repressed in unstressed cells and repression appears to be determined by two mechanisms. The first, illustrated here, is through activation by HSV-1 infection of the gene encoding GADD45γ. The second mechanism requires constitutively active expression of LGP2 and HDAC4.IMPORTANCE Previous studies from our laboratory reported that knockout of some innate immunity genes was associated with increases in the expression of overlapping networks of genes and significant loss of the ability to support the replication of HSV-1; knockout of other genes was associated with decreases in the expression of overlapping networks of genes and had no effect on virus replication. In this report, we document that depletion of GADD45γ reduced virus yields concurrently with significant upregulation of the expression of a cluster of innate immunity genes comprising IFI16, IFIT1, MDA5, and RIG-I. This report differs from the preceding study in an important respect; i.e., the preceding study found no evidence to support the hypothesis that HSV-1 maintained adequate levels of LGP2 or HDAC4 to block upregulation of the cluster of innate immunity genes. We show that HSV-1 causes upregulation of the GADD45γ gene to prevent the upregulation of innate immunity genes.
Asunto(s)
Proteínas de Ciclo Celular/inmunología , Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , Inmunidad Innata/inmunología , Proteínas Nucleares/inmunología , Animales , Chlorocebus aethiops , Histona Desacetilasas/inmunología , Proteínas de la Membrana/inmunología , ARN Helicasas/inmunología , ARN Interferente Pequeño/inmunología , Células Vero , Replicación Viral/inmunologíaRESUMEN
Dengue is a severe emerging arthropod borne viral disease occurring globally. Around two fifths of the world's population, or up to 3.9 billion people, are at a risk of dengue infection. Infection induces a life-long protective immunity to the homologous serotype but confers only partial and transient protection against subsequent infection caused by other serotypes. Thus, there is a need for a vaccine which is capable of providing a life- long protection against all the serotypes of dengue virus. In our study, comparative genomics of Dengue virus (DENV) was conducted to explore potential candidates for novel vaccine targets. From our analysis we successfully found 100% conserved epitopes in Envelope protein (RCPTQGE); NS3 (SAAQRRGR, PGTSGSPI); NS4A (QRTPQDNQL); NS4B (LQAKATREAQKRA) and NS5 proteins (QRGSGQV) in all DENV serotypes. Some serotype specific conserved motifs were also found in NS1, NS5, Capsid, PrM and Envelope proteins. Using comparative genomics and immunoinformatics approach, we could find conserved epitopes which can be explored as peptide vaccine candidates to combat dengue worldwide. Serotype specific epitopes can also be exploited for rapid diagnostics. All ten proteins are explored to find the conserved epitopes in DENV serotypes, thus making it the most extensively studied viral genome so far.