RESUMEN
This post-hoc analysis evaluated candidate biomarkers of long-term efficacy of subcutaneous interferon beta-1a (sc IFN ß-1a) in REFLEX/REFLEXION studies of clinically isolated syndrome. Samples from 507 REFLEX and 287 REFLEXION study participants were analyzed. All investigated biomarkers were significantly upregulated 1.5-4-fold in response to sc IFN ß-1a treatment versus baseline (p ≤ 0.008). The validity of MX1, 2'5'OAS, and IL-1RA as biomarkers of response to sc IFN ß-1a was confirmed in this large patient cohort, with biomarkers consistently upregulated in a dose-dependent manner. Neopterin, TRAIL, and IP-10 were confirmed as biomarkers associated with long-term sc IFN ß-1a treatment efficacy over 5 years.
Asunto(s)
Interferón beta-1a/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , 2',5'-Oligoadenilato Sintetasa/biosíntesis , 2',5'-Oligoadenilato Sintetasa/sangre , 2',5'-Oligoadenilato Sintetasa/genética , Biomarcadores , Quimiocina CXCL10/biosíntesis , Quimiocina CXCL10/sangre , Quimiocina CXCL10/genética , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Estudios de Seguimiento , Humanos , Inyecciones Subcutáneas , Interferón beta-1a/administración & dosificación , Interferón beta-1a/farmacocinética , Proteína Antagonista del Receptor de Interleucina 1/biosíntesis , Proteína Antagonista del Receptor de Interleucina 1/sangre , Proteína Antagonista del Receptor de Interleucina 1/genética , Estudios Multicéntricos como Asunto , Esclerosis Múltiple/sangre , Proteínas de Resistencia a Mixovirus/biosíntesis , Proteínas de Resistencia a Mixovirus/sangre , Proteínas de Resistencia a Mixovirus/genética , Neopterin/biosíntesis , Neopterin/sangre , Neopterin/genética , Ensayos Clínicos Controlados Aleatorios como Asunto/estadística & datos numéricos , Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Ligando Inductor de Apoptosis Relacionado con TNF/sangre , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Regulación hacia ArribaRESUMEN
High expression of 2',5'-oligoadenylate synthetase 1 (OAS1), which adds AMP residues in 2',5' linkage to a variety of substrates, is observed in many cancers as a part of the interferon-related DNA damage resistance signature (IRDS). Poly(ADP-ribose) (PAR) is rapidly synthesized from NAD+ at sites of DNA damage to facilitate repair, but excessive PAR synthesis due to extensive DNA damage results in cell death by energy depletion and/or activation of PAR-dependent programmed cell death pathways. We find that OAS1 adds AMP residues in 2',5' linkage to PAR, inhibiting its synthesis in vitro and reducing its accumulation in cells. Increased OAS1 expression substantially improves cell viability following DNA-damaging treatments that stimulate PAR synthesis during DNA repair. We conclude that high expression of OAS1 in cancer cells promotes their ability to survive DNA damage by attenuating PAR synthesis and thus preventing cell death.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/biosíntesis , Daño del ADN , Regulación Enzimológica de la Expresión Génica , Poli ADP Ribosilación , 2',5'-Oligoadenilato Sintetasa/genética , Adenosina Monofosfato/genética , Adenosina Monofosfato/metabolismo , Muerte Celular , Línea Celular Transformada , HumanosRESUMEN
The most notable effect of prenatal Zika virus (ZIKV) infection is severe microcephaly. ZIKV has a selective tropism for neural progenitor cells; however, it is not clear what role the immune cells of the brain, microglia, may have in mitigating or exacerbating neuronal cell death following ZIKV infection. We cultured hippocampal and cortical neural cells from neonatal rat pups and infected them with ZIKV at various multiplicities of infection (MOI). We found that the neuroimmune response to ZIKV infection is composed of both pro-inflammatory and type I interferon responses and is largely dependent upon the viral dose.
Asunto(s)
Células-Madre Neurales/virología , Infección por el Virus Zika/inmunología , Virus Zika/patogenicidad , 2',5'-Oligoadenilato Sintetasa/biosíntesis , Animales , Animales Recién Nacidos , Células Cultivadas , Corteza Cerebral/citología , Femenino , Hipocampo/citología , Interferón beta/biosíntesis , Interleucina-6/biosíntesis , Masculino , Microglía/inmunología , Proteínas de Resistencia a Mixovirus/biosíntesis , Células-Madre Neurales/inmunología , Células-Madre Neurales/metabolismo , Ratas , Tropismo ViralRESUMEN
The innate immune system is a broad collection of critical intra- and extra-cellular processes that limit the infectivity of diverse pathogens. The 2'-5'-oligoadenylate synthetase (OAS) family of enzymes are important sensors of cytosolic double-stranded RNA (dsRNA) that play a critical role in limiting viral infection by activating the latent ribonuclease (RNase L) to halt viral replication and establish an antiviral state. Attesting to the importance of the OAS/RNase L pathway, diverse viruses have developed numerous distinct strategies to evade the effects of OAS activation. How OAS proteins are regulated by viral or cellular RNAs is not fully understood but several recent studies have provided important new insights into the molecular mechanisms of OAS activation by dsRNA. Other studies have revealed unanticipated features of RNA sequence and structure that strongly enhance activation of at least one OAS family member. While these discoveries represent important advances, they also underscore the fact that much remains to be learned about RNA-mediated regulation of the OAS/RNase L pathway. In particular, defining the full complement of RNA molecular signatures that activate OAS is essential to our understanding of how these proteins maximize their protective role against pathogens while still accurately discriminating host molecules to avoid inadvertent activation by cellular RNAs. A more complete knowledge of OAS regulation may also serve as a foundation for the development of novel antiviral therapeutic strategies and lead the way to a deeper understanding of currently unappreciated cellular functions of the OAS/RNase L pathway in the absence of infection. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Translation > Translation Regulation.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/biosíntesis , Activadores de Enzimas/metabolismo , Regulación de la Expresión Génica , Factores Inmunológicos/biosíntesis , ARN Bicatenario/metabolismo , Endorribonucleasas/biosíntesisRESUMEN
2', 5'-Oligoadenylate synthetase-lilke (OASL) protein is an atypical oligoadenylate synthetase (OAS) family member, which possesses antiviral activity but lacks 2', 5'-oligoadenylate synthetase activity. Here, a novel variant of porcine OASL (pOASL2) was identified through RT-PCR amplification. This gene is distinguishable from the previously described wild-type porcine OASL (pOASL1). The gene appears to be derived from a truncation of exon 4 plus 8 nucleotides of exon 5 with a premature termination, measuring only 633 bp in length, although its position corresponds to that of pOASL1. Given this novel gene appears to be a variant of pOASL, we assayed for antiviral activity of the protein. We demonstrated that pOASL2 could inhibit Japanese encephalitis virus (JEV) proliferation as well as pOASL1 in a transient overexpression assay of pOASL1 and pOASL2 in PK-15 and Vero cells. In addition to JEV, pOASL1 and pOASL2 also decreased the proliferations of Porcine reproductive and respiratory syndrome virus (PRRSV) and vesicular stomatitis virus (VSV), but did not exhibit antiviral activity against pseudorabies virus (PRV). Structural analysis showed that the pOASL2 gene retained only the first three exons at the 5'-. To investigate the role of the αN4 helix in pOASL in antiviral responses like that in hOASL, we mutated key residues in the anchor domain of the αN4 helix in pOASL2, based on the domain's location in hOASL. However, the antiviral activity of pOASL2 was not affected. Thus, the αN4 helix of pOASL likely does not play a significant role in its antiviral activity. In conclusion, pOASL2 acts as a new splice isoform of pOASL that plays a role in resistance to infection of several kinds of RNA viruses.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/farmacología , Empalme Alternativo , Antivirales/farmacología , Virus de la Encefalitis Japonesa (Especie)/efectos de los fármacos , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Vesiculovirus/efectos de los fármacos , 2',5'-Oligoadenilato Sintetasa/biosíntesis , 2',5'-Oligoadenilato Sintetasa/química , 2',5'-Oligoadenilato Sintetasa/genética , Secuencia de Aminoácidos , Animales , Antivirales/química , Línea Celular , Chlorocebus aethiops , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/crecimiento & desarrollo , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Células Epiteliales/virología , Exones , Herpesvirus Suido 1/efectos de los fármacos , Herpesvirus Suido 1/genética , Herpesvirus Suido 1/crecimiento & desarrollo , Herpesvirus Suido 1/metabolismo , Isoenzimas/biosíntesis , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/farmacología , Riñón/efectos de los fármacos , Riñón/patología , Riñón/virología , Sistemas de Lectura Abierta , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/crecimiento & desarrollo , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Porcinos , Células Vero , Vesiculovirus/genética , Vesiculovirus/crecimiento & desarrollo , Vesiculovirus/metabolismoRESUMEN
HIV-associated neurocognitive disorders are common in HIV-infected individuals, even in the combination antiretroviral therapy (c-ART) era. Several mechanisms are involved in neuronal damage, including chronic inflammation immune activation. Mammalian 2'-5'-oligoadenylate synthetase (OAS) genes are produced in response to interferon (IFN), mainly by monocytes, and exert their antiviral functions by activation of RNase L that degrades viral and cellular RNAs. In this study, we aimed at exploring OAS gene family RNA expression in simian immunodeficiency virus encephalitis (SIVE), in HIV-associated neurocognitive disorders (HAND), and in HIV-associate dementia (HAD). We analyzed three microarray datasets obtained from the NCBI in order to assess the expression levels of OAS gene family network in brain biopsies of macaques with SIVE vs uninfected animals, as well as post-mortem brain of individuals with HAND (on or off ART) vs uninfected controls and three brain regions of HIV-infected individuals with both neurocognitive impairment (HAD) and encephalitis (HIVE). All OAS genes were upregulated both in SIVE and in HAND. OAS expression was significantly higher in high-viremic individuals; increased expression levels persisted in cART subjects when compared to healthy controls. OAS gene network analysis showed that several genes belonging to the type I IFN pathway, especially CXCL10 and IFIT3, were similarly upregulated in SIVE/HAND. Furthermore, we identified a significant upregulation of OAS gene family RNA expression in basal ganglia, white matter, and frontal cortex of HIV-1, HAD, and HAD/HIVE patients compared to healthy subjects. OAS gene family expression is increased in brain sections from individuals with HAND, HAD, and HIVE as well as macaques with SIVE. OAS family expression is likely to be induced by IFN as a consequence of viral replication in the CNS. Its long-term upregulation may contribute to the chronic inflammatory status and neurocognitive impairment we still observe in virologically suppressed individuals on c-ART.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/biosíntesis , 2',5'-Oligoadenilato Sintetasa/genética , Estudios de Asociación Genética/métodos , Infecciones por VIH/genética , Trastornos Neurocognitivos/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Animales , Bases de Datos Genéticas , Expresión Génica , Redes Reguladoras de Genes/genética , Infecciones por VIH/complicaciones , Infecciones por VIH/metabolismo , Hipocampo/metabolismo , Humanos , Macaca mulatta , Masculino , Trastornos Neurocognitivos/etiología , Trastornos Neurocognitivos/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/complicaciones , Síndrome de Inmunodeficiencia Adquirida del Simio/metabolismoRESUMEN
Sjögren's syndrome (SS) is a common, autoimmune exocrinopathy distinguished by keratoconjunctivitis sicca and xerostomia. Patients frequently develop serious complications including lymphoma, pulmonary dysfunction, neuropathy, vasculitis, and debilitating fatigue. Dysregulation of type I interferon (IFN) pathway is a prominent feature of SS and is correlated with increased autoantibody titers and disease severity. To identify genetic determinants of IFN pathway dysregulation in SS, we performed cis-expression quantitative trait locus (eQTL) analyses focusing on differentially expressed type I IFN-inducible transcripts identified through a transcriptome profiling study. Multiple cis-eQTLs were associated with transcript levels of 2'-5'-oligoadenylate synthetase 1 (OAS1) peaking at rs10774671 (PeQTL = 6.05 × 10-14). Association of rs10774671 with SS susceptibility was identified and confirmed through meta-analysis of two independent cohorts (Pmeta = 2.59 × 10-9; odds ratio = 0.75; 95% confidence interval = 0.66-0.86). The risk allele of rs10774671 shifts splicing of OAS1 from production of the p46 isoform to multiple alternative transcripts, including p42, p48, and p44. We found that the isoforms were differentially expressed within each genotype in controls and patients with and without autoantibodies. Furthermore, our results showed that the three alternatively spliced isoforms lacked translational response to type I IFN stimulation. The p48 and p44 isoforms also had impaired protein expression governed by the 3' end of the transcripts. The SS risk allele of rs10774671 has been shown by others to be associated with reduced OAS1 enzymatic activity and ability to clear viral infections, as well as reduced responsiveness to IFN treatment. Our results establish OAS1 as a risk locus for SS and support a potential role for defective viral clearance due to altered IFN response as a genetic pathophysiological basis of this complex autoimmune disease.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , Interferón Tipo I/genética , Sitios de Carácter Cuantitativo/genética , Síndrome de Sjögren/genética , 2',5'-Oligoadenilato Sintetasa/biosíntesis , Alelos , Empalme Alternativo/genética , Femenino , Regulación de la Expresión Génica , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Interferón Tipo I/metabolismo , Masculino , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/patología , Virosis/genética , Virosis/virologíaRESUMEN
The 2', 5'-oligoadenylate synthetases (OAS) are antiviral proteins and several isoforms have been identified as flavivirus-resistance biomarkers in human and mouse. The expression kinetics and antiviral functions of porcine OAS family (OAS1, OAS2, and OASL) in PK-15 cells following infection by Japanese encephalitis virus (JEV) were evaluated in the present study. The endogenous expression of the three OAS genes was efficiently induced by IFN-α treatment in PK-15 cells. However, expression of pOAS1 and pOAS2 responded more quickly than pOASL. Infection by JEV also induced the expression of the pOAS isoforms, but at a significantly lower level than that observed following IFN-α stimulation. Transient overexpression of pOASL and pOAS1 inhibited JEV replication more efficiently than OAS2 overexpression. Interestingly, knockdown of pOAS2 expression by siRNA treatment led to the highest increase in JEV multiplication. Co-silencing of RNase L and each pOAS revealed that the anti-JEV function of pOAS1 and pOAS2 were RNase L dependent, while the antiviral activity of pOASL was not. In conclusion, all pOAS isoforms play a significant role in the response to JEV infection, and are differentially induced by different stimuli. The alternative pathways of antiviral activity stimulated by OASL require further study.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Virus de la Encefalitis Japonesa (Especie)/inmunología , Virus de la Encefalitis Japonesa (Especie)/fisiología , Interacciones Huésped-Patógeno , Replicación Viral , 2',5'-Oligoadenilato Sintetasa/biosíntesis , 2',5'-Oligoadenilato Sintetasa/genética , Animales , Línea Celular , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Interferón-alfa/metabolismo , PorcinosRESUMEN
Lipopolysaccharide (LPS) is a critical component of the outer membrane of Gram—negative bacteria. Many cellular signals that are activated by Gram—negative bacteria are initiated by LPS. LPS triggers not only inflammatory responses, but also activates pro—apoptotic signals in a series of human cell types. However, there is relatively minimal data on the microRNA—dependent mechanism(s) of LPS—induced functional activity in osteoblast cells. CCK—8 assay and flow cytometry were used to measure cell viability and apoptosis, respectively. RT—PCR and western blot were performed to determine the mRNA and protein expression in osteoblast cells. In this study, we found that LPS triggered apoptosis in osteoblastic hFOB1.19 cells and induced a low expression of the miRNA—21. Furthermore, through the gene microarray technique, OAS1 was screened and later confirmed to be the target gene which was up—regulated in response to the low expression of miRNA—21. Knockdown of OAS1 by specific siRNAs significantly rescued the LPS—induced hFOB1.19 cell apoptosis. Our data suggest that LPS induces low expression of miRNA—21which consequently causes the up—regulation of the downstream gene OAS1 and eventually triggers apoptosis in hFOB1.19 cells. Knockdown of OAS1 rescues LPS—induced cell death and thus may be a promising therapeutic strategy for orthopedic diseases.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/biosíntesis , Apoptosis/genética , Regulación de la Expresión Génica/genética , Lipopolisacáridos , MicroARNs/biosíntesis , Osteoblastos/fisiología , 2',5'-Oligoadenilato Sintetasa/genética , Línea Celular , Supervivencia Celular , Humanos , MicroARNs/genética , Osteoblastos/citología , Interferencia de ARN , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia ArribaRESUMEN
BACKGROUND: In viral disease, infection is controlled at the cellular level by type I interferon (IFN-I), but dengue virus (DENV) has the ability to inhibit this response. Type III interferon, also known as lambda IFN (IFN-III or IFN-λ), is a complementary pathway to the antiviral response by IFN-I. This work analyzed the IFN-λ (IFN-III) mediated antiviral response against DENV serotype 2 (DENV-2) infection. METHODS: Dengue fever patients were sampled to determine their IFN-λ levels by ELISA. To study the IFN-λ response during DENV infection we selected the epithelial cell line C33-A, and we demonstrated that it is permissive to DENV-2 infection. The effect of IFN-λ on virus replication was determined in these cells, in parallel to the expression of IFN-stimulated genes (ISGs), and Suppressor of Cytokine Signaling (SOCS), genes measured by RT-qPCR. RESULTS: We found increased (~1.8 times) serological IFN-λ in dengue fever patients compared to healthy blood donors. IFN-λ inhibited DENV-2 replication in a dose-dependent manner in vitro. The reduction of viral titer corresponded with increased ISG mRNA levels (MX1 and OAS1), with the highest inhibition occurring at ISG's peak expression. Presence of IFN-negative regulators, SOCS1 and SOCS3, during DENV-2 infection was associated with reduced IFN-λ1 expression. CONCLUSIONS: Evidence described here suggests that IFN-λ is a good candidate inhibitor of viral replication in dengue infection. Mechanisms for the cellular and organismal interplay between DENV and IFN- λ need to be further studied as they could provide insights into strategies to treat this disease. Furthermore, we report a novel epithelial model to study dengue infection in vitro.
Asunto(s)
Antivirales/metabolismo , Virus del Dengue/inmunología , Virus del Dengue/fisiología , Células Epiteliales/inmunología , Células Epiteliales/virología , Interleucinas/metabolismo , Replicación Viral/efectos de los fármacos , 2',5'-Oligoadenilato Sintetasa/biosíntesis , 2',5'-Oligoadenilato Sintetasa/genética , Antivirales/sangre , Perfilación de la Expresión Génica , Humanos , Interferones , Interleucinas/sangre , Proteínas de Resistencia a Mixovirus/biosíntesis , Proteínas de Resistencia a Mixovirus/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Carga ViralRESUMEN
The Lactobacillus gasseri SBT2055 (LG2055) is a probiotic lactic acid bacterium with properties such as bile tolerance and ability to improve the intestinal environment. In this study, we established that the oral administration of LG2055 exhibits efficacy to protect mice infected with the influenza virus A/PR8. The body weight losses were lower with the LG2055 administration after the PR8 virus infection. At 5 days after the infection, the virus titer was significantly decreased as was the amount of produced IL-6 in the lung tissue, the number of total cells in the bronchoalveolar lavage fluid was reduced by the LG2055 administration. The expression of the Mx1 and Oas1a genes, critical for the viral clearance in the lung tissues was increased by the pre-treatment with LG2055. These findings suggest that the LG2055 administration is effective for the protection against influenza A virus infection by the down-regulation of viral replication through the induction of antiviral genes expression.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/inmunología , Lactobacillus/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Probióticos/uso terapéutico , Replicación Viral/genética , 2',5'-Oligoadenilato Sintetasa/biosíntesis , Animales , Líquido del Lavado Bronquioalveolar/citología , Línea Celular , Inflamación/inmunología , Inflamación/microbiología , Inflamación/virología , Subtipo H1N1 del Virus de la Influenza A/fisiología , Interferón beta/biosíntesis , Interferón beta/genética , Interleucina-6/biosíntesis , Pulmón/inmunología , Pulmón/virología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Resistencia a Mixovirus/biosíntesis , Proteínas de Resistencia a Mixovirus/genética , Infecciones por Orthomyxoviridae/virología , ARN Mensajero/biosíntesis , Factor de Transcripción STAT2/biosíntesisRESUMEN
The efficient regulation of intestinal immune responses is critical to colon health. Viruses, for example noraviruses, are key pathogens of the intestine. The lambda interferons (comprising three ligands: IFNL1, L2 and L3 - the so-called "Type III" interferons) constitute the most recently discovered IFN family and are known to be important in intestinal anti-viral defense. A fourth family member, IFNL4, was recently described. Expression of the IFN-lambda receptor is restricted to epithelial and immune cells; together, these ligands and their receptor represent an important anti-viral and immunoregulatory component of the immune/epithelial inteface. We investigated control of IFNL1 expression in human colon epithelial cells. We used the TLR3 agonist poly I:C to drive expression of IFNL1 in SW480 cells, and small interfering RNA (siRNA) to knockdown target transcription factors. We identified ZEB1 and BLIMP-1 as transcription factors that strongly inhibited IFNL1 expression in SW480 cells. Interestingly, while BLIMP-1 inhibited both type-III and type-I interferons (IFN-ß), the inhibitory action of ZEB1 was specific for IFNL1. We also defined the NF-κB family member, p65 as a key activator of IFNL1 and NF-κB p50 as a key inhibitor. Finally, we demonstrated that siRNA targeting of ZEB1 or NF-κB p50 resulted in a significant elevation of secreted IFN-λ1 protein and expression of the anti-viral gene OAS1, while knockdown of p65 inhibited these events. Our data provide insight to the regulation of IFNL1 expression in the human colon and suggest novel therapeutic approaches to elevate IFNλ-1 protein where required.
Asunto(s)
Colon/metabolismo , Células Epiteliales/metabolismo , Interleucinas/biosíntesis , 2',5'-Oligoadenilato Sintetasa/biosíntesis , Línea Celular Tumoral , Colon/citología , Células HT29 , Proteínas de Homeodominio/genética , Humanos , Interferones , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Poli I-C/farmacología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Represoras/genética , Receptor Toll-Like 3/agonistas , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factores de Transcripción/genética , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
The effects of Ridostin on the transcription of IFN family genes in human fibroblasts and lymphocytes were studied by quantitative real-time PCR. The degree of gene induction by Ridostin was most pronounced in fibroblasts, and was significantly higher than the induction by Kagocel: transcription of IFN-ß, oligoadenylate synthetase, and double-stranded RNA-dependent protein kinase genes increased by about 2000, 100, and 20 times, respectively. In lymphocytes, Ridostin also activated a wide variety of IFN family genes, including genes of IFN-ß, IFN-γ, and IFN-dependent enzymes, but this induction was less pronounced than in the fibroblasts. It was shown that gene response in lymphocyte from a child with cancer is reduced in comparison with that of adult healthy participant. Ridostin, and even more so Reaferon up-regulated activities of ß-actin, glycerophosphate dehydrogenase, and ß2-microglobulin genes, thus making impossible or limiting their use as constitutive stable reference genes (standards) in PCR-assays of IFN and their inductors.
Asunto(s)
Inductores de Interferón/farmacología , Interferones/biosíntesis , ARN Bicatenario/farmacología , ARN de Hongos/farmacología , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , 2',5'-Oligoadenilato Sintetasa/biosíntesis , 2',5'-Oligoadenilato Sintetasa/genética , Actinas/biosíntesis , Actinas/genética , Adulto , Antivirales/farmacología , Línea Celular , Niño , Fibroblastos/metabolismo , Glicerolfosfato Deshidrogenasa/biosíntesis , Glicerolfosfato Deshidrogenasa/genética , Gosipol/análogos & derivados , Gosipol/farmacología , Humanos , Interferón alfa-2 , Interferón-alfa/farmacología , Interferón beta/biosíntesis , Interferón beta/genética , Interferón gamma/biosíntesis , Interferón gamma/genética , Interferones/genética , Linfocitos/metabolismo , Virus Maus Elberfeld/efectos de los fármacos , Proteínas Recombinantes/farmacología , Microglobulina beta-2/biosíntesis , Microglobulina beta-2/genética , eIF-2 Quinasa/biosíntesis , eIF-2 Quinasa/genéticaRESUMEN
The type I interferons (IFN-Is) are critical not only in early viral control but also in prolonged T-cell immune responses. However, chronic viral infections such as those of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in humans and lymphocytic choriomeningitis virus (LCMV) in mice overcome this early IFN-I barrier and induce viral persistence and exhaustion of T-cell function. Although various T-cell-intrinsic and -extrinsic factors are known to contribute to induction of chronic conditions, the roles of IFN-I negative regulators in chronic viral infections have been largely unexplored. Herein, we explored whether 2'-5' oligoadenylate synthetase-like 1 (OASL1), a recently defined IFN-I negative regulator, plays a key role in the virus-specific T-cell response and viral defense against chronic LCMV. To this end, we infected Oasl1 knockout and wild-type mice with LCMV CL-13 (a chronic virus) and monitored T-cell responses, serum cytokine levels, and viral titers. LCMV CL-13-infected Oasl1 KO mice displayed a sustained level of serum IFN-I, which was primarily produced by splenic plasmacytoid dendritic cells, during the very early phase of infection (2-3 days post-infection). Oasl1 deficiency also led to the accelerated elimination of viremia and induction of a functional antiviral CD8 T-cell response, which critically depended on IFN-I receptor signaling. Together, these results demonstrate that OASL1-mediated negative regulation of IFN-I production at an early phase of infection permits viral persistence and suppresses T-cell function, suggesting that IFN-I negative regulators, including OASL1, could be exciting new targets for preventing chronic viral infection.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Linfocitos T CD8-positivos/inmunología , Regulación hacia Abajo , Interferón Tipo I/metabolismo , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Receptor de Interferón alfa y beta/metabolismo , 2',5'-Oligoadenilato Sintetasa/biosíntesis , 2',5'-Oligoadenilato Sintetasa/genética , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Resistencia a la Enfermedad , Femenino , Inmunidad Innata , Factor 7 Regulador del Interferón/biosíntesis , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Interferón Tipo I/sangre , Interferón Tipo I/genética , Coriomeningitis Linfocítica/sangre , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/virología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta/antagonistas & inhibidores , Receptor de Interferón alfa y beta/genética , Transducción de Señal , Viremia/sangre , Viremia/inmunología , Viremia/metabolismo , Viremia/virologíaRESUMEN
Based on investigations of liver biopsy material, certain cellular genes have been implicated as correlates of success or failure to interferon alpha-ribavirin (IFN/RBV) therapy against hepatitis C. The current study aimed at determining whether expression of host genes thought to be relevant to HCV replication in the liver would be correlated with HCV infection status in peripheral blood mononuclear cells (PBMCs) and also with patient responsiveness to IFN/RBV treatment. Therefore, PBMCs from patients with chronic hepatitis C responding (n = 35) or not (n = 49) to IFN/RBV and from healthy controls (n = 15) were evaluated for HCV RNA load and cellular gene expression. Non-responders had 3- to 10-fold higher basal levels of interleukin (IL)-8, IFN-stimulated gene 15 (ISG15), 2',5'-oligoadenylate synthetase (OAS), and Toll-like receptors (TLR)-4, -5, and -7 compared to responders. Non-responders with similar post-treatment follow-ups as responders persistently expressed 6- to 20-fold greater levels of IL-8, ISG15, and OAS after therapy. Higher expression of IFN-α, IFN-γ, and IFN-λ was found in PBMCs of individuals achieving sustained virological response, either before or after therapy. Pre-treatment HCV RNA loads in PBMCs of non-responders were significantly higher (P = 0.016) than those of responders. In conclusion, the data indicate that immune cells of responders and non-responders to IFN/RBV therapy exhibited significantly different virological and host gene expression profiles. Elevated baseline HCV loads and TLR-4, -5, and -7 levels, and persistently high levels of IL-8, ISG15, and OAS were correlated with IFN non-responsiveness. The results warrant further investigations on the utilization of PBMCs for predicting success or failure to IFN-based therapies.
Asunto(s)
Antivirales/uso terapéutico , Hepacivirus/aislamiento & purificación , Hepatitis C Crónica/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Linfocitos/inmunología , Ribavirina/uso terapéutico , Carga Viral , 2',5'-Oligoadenilato Sintetasa/biosíntesis , Adolescente , Adulto , Anciano , Niño , Citocinas/biosíntesis , Femenino , Hepacivirus/inmunología , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , ARN Viral/sangre , Receptores Toll-Like/biosíntesis , Resultado del Tratamiento , Ubiquitinas/biosíntesis , Adulto JovenRESUMEN
The aim of the study was to determine the ability of several chemically different antiviral substances to induce the expression of interferon α(IFNα), PKR, OAS1a and RNAse L genes in the rat liver. The investigated substances included Amizon, Altabor and Proteflasid, which are already used in practical medicine, and 3',7-dimethylquercetin extracted from Proteflasid, the mixture of synthesized trimethyl- and tetramethylquercetin and Sialospecific lectin from persimmon, which are at the stage of preclinical trial and experimental research respectively. The content of corresponding mRNAs in total RNA was detected with the help of reverse transcription and polymerase chain reaction in real time. The results have shown that all investigated substances induce the expression of genes α, PKR, OAS RNAse L in specific manner. The combination of 3',7-dimethylquercetin + lectin from persimmon had the highest stimulating effect exceeding the effect of each component of the mixture and the influence of Heberon (recombinant IFNα2b) and PolyI-polyC as the standard inducers of IFNα and its target genes. The ability of all substances to specifically induce the expression of IFNa and its target genes, the absence of correlation between the levels of IFNα and its target genes expression as well as between target genes themselves indicate that the mechanism of antiviral activity of the investigated substances is connected not only with up-regulation of IFNα and potential IFNα mediated effects.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/biosíntesis , Antivirales/administración & dosificación , Endorribonucleasas/biosíntesis , Expresión Génica/efectos de los fármacos , Interferón-alfa/biosíntesis , eIF-2 Quinasa/biosíntesis , 2',5'-Oligoadenilato Sintetasa/genética , Animales , Diospyros/química , Endorribonucleasas/genética , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Interferón-alfa/genética , Lectinas/administración & dosificación , Lectinas/aislamiento & purificación , Hígado/efectos de los fármacos , Hígado/metabolismo , Poli I-C/administración & dosificación , Reacción en Cadena de la Polimerasa , Piridinas/administración & dosificación , Compuestos de Piridinio , Quercetina/administración & dosificación , Quercetina/análogos & derivados , Quercetina/síntesis química , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Proteínas Recombinantes/administración & dosificación , Taninos/administración & dosificación , Taninos/aislamiento & purificación , eIF-2 Quinasa/genéticaRESUMEN
Human 2',5'-oligoadenylate synthetase 3 (OAS3) exerts antiviral effect against alphaviruses including Chikungunya virus (CHIKV) by inhibiting viral RNA accumulation. Here, we identified a CHIKV variant exhibiting a remarkable resistance to the antiviral action of OAS3 in human epithelial HeLa cells. Using a molecular clone of CHIKV with Renilla luciferase inserted as a reporter gene in the non-structural region, we demonstrated that a single glutamine-to-lysine amino acid change at position 166 of the envelope E2 glycoprotein restores CHIKV replication in OAS3 expressing HeLa cells. Viral entry assays showed that CHIKV with a lysine at position E2-166 was more efficient at entering the replicative pathway. The E2-E166K substitution promotes a greater efficiency of CHIKV replication in human myoblasts leading to severe apoptosis through a more robust activation of the PKR pathway. These observations provide a new insight into the role of E2 into the pathogenicity of CHIKV in human cells.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/biosíntesis , Virus Chikungunya/fisiología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Internalización del Virus , 2',5'-Oligoadenilato Sintetasa/inmunología , Animales , Apoptosis , Fusión Artificial Génica , Virus Chikungunya/genética , Virus Chikungunya/crecimiento & desarrollo , Virus Chikungunya/inmunología , Genes Reporteros , Células HeLa , Humanos , Luciferasas/análisis , Luciferasas/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mioblastos/fisiología , Mioblastos/virología , Renilla/enzimologíaRESUMEN
Double-stranded RNA (dsRNA) can induce antiviral enzyme 2',5'-oligoadenylate synthetase (2'5'AS) expression and activate latent 2'5'AS. Our previous data have shown pancreatic ß cells are sensitive to dsRNA-induced 2'5'AS expression, and constitutive high basal 2'5'AS expression is associated with susceptibility to developing type 1 diabetes (T1D), a disease due to pancreatic ß cell loss. Here we report that in vitro transcribed human insulin mRNA induces the activation of human OAS gene promoter sequences, and specifically and dose-dependently induces 2'5'AS expression in murine pancreatic ßTC3 cells. Over-expression of dsRNA receptor retinoic acid-inducible gene-1 enhances insulin mRNA-induced 2'5'AS expression. In vitro transcribed insulin and other mRNAs, as well as total cellular RNAs, activate latent 2'5'AS in vitro with activation ability likely associated with the sequence and length of individual mRNAs or the sample source of total cellular RNA. Insulin mRNA does not show any specificity to activate 2'5'AS, but total cellular RNA from ßTC3 cells has high activation ability. Constitutive 2'5'AS expression in ßTC3 cells leads to cell proliferation inhibition and apoptosis. Our study suggests the possibility of cellular RNA-regulated 2'5'AS expression and activation, and the potential risk of high insulin gene transcription in pancreatic ß cells, and may help explain genetic predisposition to T1D associated with INS VNTR class I alleles.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/biosíntesis , Diabetes Mellitus Tipo 1/genética , Células Secretoras de Insulina/metabolismo , Insulina/genética , ARN Bicatenario/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/biosíntesis , Predisposición Genética a la Enfermedad , Células HEK293 , Células HeLa , Humanos , Células Secretoras de Insulina/enzimología , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos BALB C , Repeticiones de Minisatélite , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Toll-Like 7/biosíntesisRESUMEN
The 2',5'-oligoadenylate synthetases (OASs) are IFN-induced antiviral proteins and are upregulated by infection of viral and some bacterial pathogens. There are at least 2 transcripts of approximately 1.8 and 2.0 kb in interferon-beta treated samples that are recognized by a probe for human OASL in Northern blot assay. By RT-PCR amplification we have isolated a previously undescribed splice variant of human OASL, named OASL d. The new variant was derived from deletion of exons 4 and 5 and encodes a protein of 384 aa residues that shares the N-terminal 219 aa residues with OASL a. Sequence analysis indicates that OASL d also contains the entire ubiquitin-like domain identified in human OASL a. OASL d was strongly induced by IFNγ in THP-1 monocytic cells and in A549 epithelial cells by interferon-beta as detected by immunoblotting assay. Ectopic expression of OASL a or OASL d, but not OASL b that shares the N-terminus with OASL a and d, partially inhibited EV71 and VSV infection. No effect against HSV-2 infection was observed. Therefore, OASL d is a novel isoform of human OASL that possesses antiviral activity against RNA viruses.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/biosíntesis , 2',5'-Oligoadenilato Sintetasa/inmunología , 2',5'-Oligoadenilato Sintetasa/metabolismo , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Exones , Eliminación de Gen , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Interferón beta/farmacología , Isoformas de Proteínas , Transfección , Células VeroRESUMEN
Antiviral effect of interferon is mediated by the expression of interferon-stimulated genes (ISGs). However, because of the difficulty in obtaining paired liver biopsies before and after interferon treatment, the key ISGs expressed in human hepatocytes and responsible for interferon-based antiviral activities in chronic hepatitis C remain illusive. Prior to a standard course of peginterferon plus ribavirin therapy, 104 patients underwent a liver biopsy. A small piece of the liver biopsy sample from each patient was submitted for ex vivo tissue culture in the presence or absence of interferon. Hepatic expression of 8 ISGs was detected by RT-PCR. The ISG expression patterns and clinicopathological variables were correlated with subsequent treatment outcomes. Multivariate logistic regression analysis showed that hepatic MxA expression (P = 0.008) and leucocyte count (P = 0.040) independently predicted the end of therapeutic virological response, while hepatic OAS1 expression (P = 0.003), genotype 1 (P = 0.002), HCV-RNA level (P = 0.007), AST/ALT ratio (P = 0.004) and leucocyte count (P = 0.034) independently predicted the sustained virological response. Immunohistochemistry analysis showed that interferon-induced OAS1 expression localized to the hepatocytes. In conclusion, hepatic MxA and OAS1 expression predicted, respectively, the end of therapeutic and sustained virological responses in interferon-based treatment of chronic hepatitis C.