RESUMEN
BACKGROUND & AIMS: Patients with chronic hepatitis C virus infection are commonly treated with interferon alfa (IFN-alpha), but the long-term response rate is poor. A region of NS5A of hepatitis C virus genotype 1 (the ISDR) has been associated with treatment outcome in some patients. NS5A binds to and inhibits PKR in vitro and inhibits IFN-alpha in human cells. We examined the effects of the NS5A protein from patients who did or did not respond to IFN-alpha to determine whether NS5A from IFN-alpha nonresponders inhibited the effects of IFN-alpha in vitro. METHODS: We cloned NS5A from patients who had well-characterized responses to IFN-alpha and expressed them in a human fibroblast cell line under the control of an inducible promoter. The NS5A expression levels were controlled, and the effects of different proteins on the protective actions of IFN-alpha against encephalomyocarditis virus were investigated. RESULTS: NS5A expression blocked the antiviral effects of IFN-alpha in human cells. This inhibition was dependent on the level of NS5A expression. Although ISDR changes gave only small differences in IFN-alpha inhibition, clones derived from a patient who did not respond to IFN-alpha and one who did respond to treatment differed greatly: the clones from a patient with response to IFN-alpha were much more inhibitory than those derived from the patient with no response. CONCLUSIONS: The inhibition of the antiviral effects of IFN-alpha by NS5A is not regulated exclusively by the ISDR, and the effects of NS5A in vitro do not correlate with treatment outcomes.
Asunto(s)
Antivirales/antagonistas & inhibidores , Interferón-alfa/antagonistas & inhibidores , Proteínas no Estructurales Virales/farmacología , Antivirales/farmacología , Antivirales/uso terapéutico , Línea Celular , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Virus de la Encefalomiocarditis/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/virología , Hepatitis C Crónica/sangre , Humanos , Interferón-alfa/farmacología , Interferón-alfa/uso terapéutico , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Transfección , Proteínas no Estructurales Virales/sangre , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismoRESUMEN
The hepatitis G virus (HGV) is a new member of the Flaviviridae family and has a genomic organization similar to that of hepatitis C virus (HCV). Protein sequence motifs are present suggesting that HGV encodes a serine proteinase, an RNA-dependent RNA polymerase and a helicase. We have cloned and expressed the putative helicase of HGV and have shown that it contains a poly (U)-stimulated NTPase activity and is able to function as a DNA helicase. Preliminary characterization of the HGV helicase activity reveals similarities with other members of the Flaviviridae, but especially with HCV, raising the possibility that HGV could be used as a surrogate virus for the development of therapies against HCV.
Asunto(s)
ADN Helicasas/genética , Flaviviridae/genética , Hepatitis Viral Humana/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Cromatografía en Capa Delgada , Clonación Molecular , ADN/análisis , ADN/metabolismo , ADN Helicasas/metabolismo , ADN Complementario/análisis , ADN Complementario/genética , ADN de Cadena Simple/análisis , ADN de Cadena Simple/metabolismo , Expresión Génica , Hepacivirus/genética , Humanos , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Viral/genética , Proteínas Recombinantes de Fusión/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
A clone of interferon-alpha-resistant (IFNr) Daudi cells retained much greater transcriptional inducibility of the (2'-5') oligoadenylate synthetase than the 6-16 gene despite the fact that the response of both genes is mediated by highly similar interferon-stimulable DNA response elements (ISRE). The primary IFN-alpha activatable transcription factor E (ISGF3) and the additional IFN-alpha-inducible ISRE-binding complex M were greatly reduced in the IFNr cells. The defect in E was in the E alpha subunit. In electrophoretic mobility-shift assays the 6-16 and (2'-5') oligoadenylate synthetase ISRE competed approximately equivalently for E and M. Moreover although active in wild-type cells the (2'-5') oligoadenylate synthetase ISRE was no more capable of conferring inducibility on a reporter gene in the IFNr cells than was the 6-16 ISRE. The contrasting response of the endogenous (2'-5') oligoadenylate synthetase and 6-16 genes in the IFNr cells is, therefore, unlikely simply to reflect the slight difference in the sequence of their ISRE. Consistent with this, in addition to the ISRE, sequences 5' to the ISRE in the (2'-5') oligoadenylate synthetase promoter appeared necessary for good induction by IFN alpha in the IFNr cells. Subtle quantitative changes in the phenotype of the IFNr cells have, however, precluded a more precise definition of the DNA element(s) involved.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/biosíntesis , 2',5'-Oligoadenilato Sintetasa/genética , Interferón-alfa/farmacología , Familia de Multigenes/efectos de los fármacos , Regiones Promotoras Genéticas , Secuencia de Bases , Línea Celular , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/metabolismo , ADN de Neoplasias/química , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Inducción Enzimática , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Prueba de Complementación Genética , Humanos , Cinética , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Factores de Tiempo , Transcripción Genética/genética , Transfección , Células Tumorales CultivadasRESUMEN
Expression of the E1A oncogene of adenovirus type 5 inhibits the response of interferon (IFN)-inducible constructs to Type I (alpha,beta) and II (gamma) IFNs in transient transfection assays. In human cell lines stably expressing E1A mRNA and protein acquisition of an antiviral state and the induction of a number of genes in response to alpha- and gamma-IFNs is inhibited. A short IFN-stimulable response element (ISRE) present in the 5' flanking region of a number of genes mediates induction by alpha- and gamma-IFNs. In cells expressing E1A there is a substantial reduction in the levels of the ISRE-binding factors E and M, inducible by alpha-IFN, and of factor G, inducible by gamma-IFN. In E1A-expressing cells the E alpha subunit of factor E is activated normally in response to alpha-IFN; the defect is in the production or activation of the E gamma subunit. The inhibitory activity of E1A is lost upon deletion of the CR1 domain. The induction of HLA class II genes by gamma-IFN, which involves a different DNA response element(s), and of beta-IFN mRNA in response to double-stranded RNA are also inhibited by E1A. An essential component(s) of a number of signalling pathways must, therefore, be subject, directly or indirectly, to inhibition by E1A.