RESUMEN
A plasmid carrying the complete genome of hepatitis B virus (HBV) inserted into the BamHI site of the pBR322 plasmid vector has been constructed. The physical map of HBV DNA established for 13 restriction endonucleases allows to conclude that the cloned DNA is similar, but not identical to the HBV DNA of ayw subtype.
Asunto(s)
ADN Viral/genética , Virus de la Hepatitis B/genética , Mapeo Cromosómico , Clonación Molecular , Enzimas de Restricción del ADN , Escherichia coli/genéticaRESUMEN
Direct expression of hepatitis B virus (HBV) surface antigen (HBsAg) gene under the control of the Escherichia coli tryptophan operon (trp) promoter has been achieved. Synthesis of HBsAg (both complete and lacking its N-terminal segment) as a part of hybrid proteins with the N-terminal portion coded by genes cat, kan or bla is controlled by the appropriate promoters, as well as by the trp promoter. The highest levels of expression, including those for direct synthesis of HBsAg, provide the accumulation of about 10(5) polypeptide molecules per bacterial cell.
Asunto(s)
Escherichia coli/genética , Genes Virales , Antígenos de Superficie de la Hepatitis B/genética , Clonación Molecular , Escherichia coli/crecimiento & desarrollo , Escherichia coli/inmunología , Regulación de la Expresión Génica , Antígenos de Superficie de la Hepatitis B/aislamiento & purificación , Operón , PlásmidosRESUMEN
Four different hybridoma clones secreting anti-HBcAg antibodies were constructed by fusing cells of the mouse myeloma line SP2/0 with lymphocytes from mice immunized with bacterially produced HBcAg. The monoclonal antibodies were immunologically characterized and used for HBcAg detection by ELISA. This monoclonal-antibody-based assay was compared with ELISA based on polyclonal human anti-HBcAg IgG for sensitivity and specificity. The monoclonal antibody reacted specifically both with the bacterially produced HBcAg and HBcAg isolated from human liver, but did not react with HBeAg. The human polyclonal antibody reacted with HBcAg, but also with HBeAg.
Asunto(s)
Anticuerpos Monoclonales , Antígenos del Núcleo de la Hepatitis B/inmunología , Proteínas Recombinantes/inmunología , Animales , Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Antígenos del Núcleo de la Hepatitis B/análisis , Antígenos del Núcleo de la Hepatitis B/genética , Humanos , Hibridomas/inmunología , Hígado/inmunología , Ratones , Ratones Endogámicos BALB C , PlásmidosRESUMEN
The gene coding for hepatitis B large envelope protein was cloned under the lac promoter in bacterial vector pUC-8 and under the ADH1 promoter in yeast expression shuttle vector pVT103-U, and expression of HBsAg in bacteria and yeast was determined. The strongest expression of large envelope protein was obtained after transformation of the protease-deficient yeast strain BJ1991. The recombinant large envelope protein did not form complex 22-nm particles and was not secreted into medium.
Asunto(s)
Escherichia coli/genética , Antígenos de Superficie de la Hepatitis B/biosíntesis , Virus de la Hepatitis B/genética , Saccharomyces cerevisiae/genética , Proteínas del Envoltorio Viral/biosíntesis , Clonación Molecular , Expresión Génica , Genes Virales , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/aislamiento & purificación , Técnicas para Inmunoenzimas , Plásmidos/genética , Transformación Bacteriana , Ultracentrifugación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
When the small form of the delta antigen (deltaAg-S) was expressed from a cDNA expression plasmid and subsequently detected by immunofluorescence, it was found localized to the nucleoli. However, if the cDNA was cotransfected with a cDNA expressing a mutated hepatitis delta virus (HDV) genome that could only replicate by using the deltaAg-S provided by the first plasmid, then most of the deltaAg-S was redistributed to the nucleoplasm, largely to specific discrete nucleoplasmic sites or speckles; this pattern was stable for at least 50 days after transfection. These speckles coincided with those detected with an antibody to SC35, an essential non-small nuclear ribonucleoprotein splicing factor. Others have shown that SC35 speckles correspond to active sites of DNA-directed transcription by RNA polymerase II and also of RNA processing. We also found, in contrast to the cotransfections with the mutant HDV and the deltaAg-S provided in trans, that cells transfected with wild-type HDV showed a variable pattern of staining. The SC35-like speckle pattern of accumulation of delta antigen deltaAg was maintained for only 6 days, after which the pattern began to change. By 18 days posttransfection, a variety of different deltaAg staining patterns were observed. This pattern of change occurs at a time when the large form of the delta antigen deltaAg-L appears and HDV RNA synthesis begins to shut down. Our studies therefore support the interpretation that HDV RNA and deltaAg-S accumulate at SC35 speckle sites in the nucleoplasm. We speculate that these may be the sites at which HDV RNA is transcribed by RNA polymerase II and/or sites of HDV RNA processing. Furthermore, when deltaAg-L, as well as other mutant deltaAg accumulate, the speckle association is disrupted, thereby stopping HDV RNA replication.
Asunto(s)
Antígenos de la Hepatitis/metabolismo , Virus de la Hepatitis Delta/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de la Hepatitis/genética , Virus de la Hepatitis Delta/genética , Virus de la Hepatitis Delta/fisiología , Antígenos de Hepatitis delta , Humanos , Datos de Secuencia Molecular , Mutagénesis , Conejos , Células Tumorales Cultivadas , Replicación ViralRESUMEN
Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-alpha) and ribavirin combination therapy. The mechanism of the IFN-alpha response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-alpha reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-alpha independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-alpha. Finally, under our conditions, selection for IFN-alpha-resistant variants did not occur.
Asunto(s)
Antivirales/farmacología , Genes Virales/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Interferón-alfa/farmacología , Replicón/efectos de los fármacos , Adaptación Fisiológica , Línea Celular , Genoma Viral , Hepacivirus/genética , Hepacivirus/fisiología , Humanos , Mutagénesis , Replicación Viral/efectos de los fármacosRESUMEN
Hepatitis delta virus (HDV) is known to express a protein termed the small delta antigen, a structural protein which is also essential for genome replication. During replication, posttranscriptional RNA editing specifically modifies some of the HDV RNA, leading to the production of an elongated form of the delta antigen, the large form, which is essential for virus assembly. The present study showed that yet another form of HDV protein is expressed during genome replication. This novel form is not produced in all infected cells, but it arises during replication in transfected cells and in infected woodchucks, and as was previously reported, patients infected with HDV do make antibodies directed against it. These findings are an indicator of the complexity of gene expression during HDV infection and replication.
Asunto(s)
Antígenos de la Hepatitis/biosíntesis , Virus de la Hepatitis Delta/metabolismo , Hígado/virología , Secuencia de Aminoácidos , Animales , Anticuerpos , Replicación del ADN , ADN Viral/metabolismo , Técnica del Anticuerpo Fluorescente , Antígenos de la Hepatitis/análisis , Antígenos de la Hepatitis/química , Hepatitis D/patología , Hepatitis D/veterinaria , Hepatitis D/virología , Virus de la Hepatitis Delta/genética , Antígenos de Hepatitis delta , Marmota , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Transfección , Replicación ViralRESUMEN
A total of 17 antibodies, raised in several nonhuman species and specific for different regions on the delta antigen (delta Ag), were used to map, via immunoprecipitation, those domains exposed on the surface of the viral ribonucleoprotein (RNP). These studies showed that the domains for the nuclear localization signal and the C-terminal extension, unique to the large form of delta Ag, are exposed. Also exposed is the C-terminal region of the small form of delta Ag. In contrast, reactivity was not found with the coiled-coil domain needed for protein dimerization. When the hepatitis delta virus (HDV) RNA was released by treatment of viral RNP with vanadyl ribonucleoside complexes, no change in the pattern of delta Ag epitope presentation was detected, consistent with the interpretation that a multimeric protein structure persists in the absence of RNA. These RNP studies have implications not only for understanding of the process of HDV assembly but also for evaluation of the immune responses of an infected host to HDV replication.
Asunto(s)
Anticuerpos Antivirales , Antígenos Virales/inmunología , Epítopos/análisis , Virus de la Hepatitis Delta/inmunología , Ribonucleoproteínas/inmunología , Proteínas Virales/inmunología , Animales , Especificidad de Anticuerpos , Antígenos Virales/biosíntesis , Antígenos Virales/aislamiento & purificación , Northern Blotting , Línea Celular , Núcleo Celular , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Cobayas , Virus de la Hepatitis Delta/metabolismo , Humanos , Marmota , Conejos , Ribonucleoproteínas/biosíntesis , Ribonucleoproteínas/aislamiento & purificación , Transfección , Proteínas Virales/biosíntesis , Proteínas Virales/aislamiento & purificación , Virión/inmunologíaRESUMEN
During the replication cycle of hepatitis delta virus (HDV), RNA editing occurs at position 1012 on the 1679-nucleotide RNA genome. This changes an A to G in the amber termination codon, UAG, of the small form of the delta antigen (delta Ag). The resultant UGG codon, tryptophan, allows the translation of a larger form of the delta Ag with a 19-amino-acid C-terminal extension. Using HDV cDNA-transfected cells, we examined the editing potential of HDV RNA mutated from G to A at 1011 on the antigenome, adjacent to normal editing site at 1012. Four procedures were used to study not only the editing of the A at 1012, but also that of the new A at 1011: (i) nucleotide sequencing, (ii) a PCR-based RNA-editing assay, (iii) immunoblot assays, and (iv) immunofluorescence. Five findings are reported. (i) Even after the mutation at 1011, editing still occurred at 1012. (ii) Site 1011 itself now acted as a novel RNA-editing site. (iii) Sites 1011 and 1012 were edited independently. (iv) At later times, both sites became edited, thereby allowing the synthesis of the large form of the delta Ag (delta Ag-L). (v) Via immunofluorescence, such double editing became apparent as a stochastic event, in that groups of cells arose in which the changes had taken place. Evaluation of these findings and of those from previous studies of the stability of the HDV genomic sequence (H.J. Netter et al., J. Virol. 69:1687-1692, 1995) supports both the recent reevaluation of HDV RNA editing as occurring on antigenomic RNA (Casey and Gerin, personal communication) and the interpretation that editing occurs via the RNA-modifying enzyme known as DRADA.