RESUMEN
The significant public health problem of Hepatitis C virus (HCV) has been partially addressed with the advent of directly acting antiviral agents (DAAs). However, the development of an effective preventative vaccine would have a significant impact on HCV incidence and would represent a major advance towards controlling and possibly eradicating HCV globally. We previously reported a genotype 1a HCV viral-like particle (VLP) vaccine that produced neutralizing antibodies (NAb) and T cell responses to HCV. To advance this approach, we produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine to produce broader immune responses. We show that this quadrivalent vaccine produces antibody and NAb responses together with strong T and B cell responses in vaccinated mice. Moreover, selective neutralizing human monoclonal antibodies (HuMAbs) targeting conserved antigenic domain B and D epitopes of the E2 protein bound strongly to the HCV VLPs, suggesting that these critical epitopes are expressed on the surface of the particles. Our findings demonstrate that a quadrivalent HCV VLP based vaccine induces broad humoral and cellular immune responses that will be necessary for protection against HCV. Such a vaccine could provide a substantial addition to highly active antiviral drugs in eliminating HCV.
Asunto(s)
Hepacivirus/inmunología , Hepatitis C/inmunología , Vacunas contra Hepatitis Viral/inmunología , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Epítopos/inmunología , Genotipo , Hepacivirus/genética , Hepatitis C/prevención & control , Anticuerpos contra la Hepatitis C/inmunología , Inmunidad Celular , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Linfocitos T/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
Liver failure associated with hepatitis C virus (HCV) accounts for a substantial portion of liver transplantation. Although current therapy helps some patients with chronic HCV infection, adverse side effects and a high relapse rate are major problems. These problems are compounded in liver transplant recipients as reinfection occurs shortly after transplantation. One approach to control reinfection is the combined use of specific antivirals together with HCV-specific antibodies. Indeed, a number of human and mouse monoclonal antibodies to conformational and linear epitopes on HCV envelope proteins are potential candidates, since they have high virus neutralization potency and are directed to epitopes conserved across diverse HCV genotypes. However, a greater understanding of the factors contributing to virus escape and the role of lipoproteins in masking virion surface domains involved in virus entry will be required to help define those protective determinants most likely to give broad protection. An approach to immune escape is potentially caused by viral infection of immune cells leading to the induction hypermutation of the immunoglobulin gene in B cells. These effects may contribute to HCV persistence and B cell lymphoproliferative diseases.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos contra la Hepatitis C/uso terapéutico , Hepatitis C/terapia , Secuencia de Aminoácidos , Linfocitos B/inmunología , Linfocitos B/virología , Epítopos , Genes env , Hepacivirus/genética , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/biosíntesis , Humanos , Datos de Secuencia Molecular , Pruebas de Neutralización , Hipermutación Somática de Inmunoglobulina , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
We determined the nucleotide and deduced amino acid sequence of the 5' terminus of the hepatitis G virus (HGV) genome from isolates of varied geographical origins. Our analysis showed that the putative 5' non-coding region (NCR) contains several blocks of highly conserved sequences that may be useful for the development of a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of HGV RNA. Overall, the degree of conservation within the 669-nucleotide (nt) 5'terminal sequence was found to range from 99.5% to 86% sequence identity. We also showed that the HGV NCR from some isolates contained conserved insertions or deletions that altered the translational reading frames at the 5'-end of the genome, resulting in different sizes of predicted polyproteins encoded by genomes of individual isolates. Specifically, the insertions/deletions affected the size of the peptide preceding the putative first envelope (E1) protein. Phylogenetic analysis of the nucleotide sequences suggested that the isolates examined can be classified into distinct groups that may be useful for studying the molecular evolution of HGV and possible relationships between isolate sequence characteristics and infection patterns.
Asunto(s)
ADN Viral/genética , Flaviviridae/química , Flaviviridae/genética , Filogenia , Proteínas E1 de Adenovirus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia Conservada/genética , Elementos Transponibles de ADN , ADN Viral/análisis , ADN Viral/química , Flaviviridae/clasificación , Eliminación de Gen , Variación Genética , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
BACKGROUND: In this study, we determined the prevalence of hepatitis G virus (HGV) infection in end-stage hepatitis C virus (HCV)-related liver disease and examined the influence of HGV coinfection on the outcome of liver transplantation. METHODS: HGV was detected by reverse transcriptase-polymerase chain reaction and Southern blotting in sera drawn from 159 patients who were known to be HCV infected before transplantation. Patients were followed up for a mean of 28.4 months after transplantation. RESULTS: Forty-one (25.3%) patients were HGV positive and the prevalence of HGV infection was similar for different HCV genotypes. Both HGV-positive and -negative groups had similar survival, recurrence rates, inflammatory activity scores, and degree of fibrosis at the time of recurrence. CONCLUSION: Infection with HGV is common in end-stage HCV-infected patients presenting for liver transplantation. It influences neither the outcome of liver transplantation nor the recurrence of hepatitis in the graft.
Asunto(s)
Flaviviridae , Hepatitis C/sangre , Hepatitis C/complicaciones , Hepatitis Viral Humana/sangre , Hepatitis Viral Humana/complicaciones , Trasplante de Hígado/efectos adversos , Adulto , Biopsia , Enfermedad Crónica , Estudios de Cohortes , Rechazo de Injerto/virología , Humanos , Hepatopatías/cirugía , Trasplante de Hígado/inmunología , Trasplante de Hígado/patología , Persona de Mediana EdadRESUMEN
Synexin (annexin VII) is a calcium-dependent, phospholipid-binding and membrane fusion protein in the annexin gene family, which forms calcium channels and may play a role in exocytotic secretion. We report here the cloning and characterization of five novel isoforms of cDNAs encoding Xenopus synexin from brain, oocyte and stage 24 cDNA libraries. The most prevalent Xenopus synexin has 1976 bp of cDNA sequence, which contains a 1539 bp open reading frame of 512 amino acids encoding a 54 kDa protein. This Xenopus protein is 6 kDa larger than the previously reported human and mouse synexins with which it shares approx. 73% identity in the C-terminal region and approx. 44% identity in the N-terminal region. Further studies with PCR revealed the molecular basis of the substantial divergence in the Xenopus synexin's N-terminal domain. The domain equivalent to the mammalian tissue-specific cassette exon occurs at a different position and is variable in size and sequence. The most interesting observation relates to the occurrence of different forms of synexin due to the varying numbers of tandem PGQM repeats that are expressed differently in different adult tissues and embryonic stages. For these reasons we have labelled this set of unique isoforms annexin VIIb, referring to mammalian forms, which lack the PGQM tandem repeats, as annexin VIIa. In spite of these differences from annexin VIIa, the form of recombinant annexin VIIb with three PGQM repeats was found to be catalytically active. We interpret these results to indicate that the actual calcium and phospholipid binding sites are conserved in Xenopus, and that the variations observed between members of the synexin gene family in the regulatory domain clearly point towards the tissue- and stage-specific roles of individual members, possibly involving the exocytotic process.
Asunto(s)
Anexina A7/biosíntesis , Evolución Biológica , Embrión no Mamífero/fisiología , Oocitos/metabolismo , ARN Mensajero/biosíntesis , Transcripción Genética , Secuencia de Aminoácidos , Animales , Anexina A7/química , Anexina A7/farmacología , Secuencia de Bases , Bovinos , Gránulos Cromafines/efectos de los fármacos , Gránulos Cromafines/fisiología , Clonación Molecular , Secuencia Conservada , ADN Complementario , Exones , Femenino , Regulación del Desarrollo de la Expresión Génica , Variación Genética , Humanos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , ARN Mensajero/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Aminoácido , XenopusRESUMEN
An RNA virus, designated hepatitis G virus (HGV), was identified from the plasma of a patient with chronic hepatitis. Extension from an immunoreactive complementary DNA clone yielded the entire genome (9392 nucleotides) encoding a polyprotein of 2873 amino acids. The virus is closely related to GB virus C (GBV-C) and distantly related to hepatitis C virus, GBV-A, and GBV-B. HGV was associated with acute and chronic hepatitis. Persistent viremia was detected for up to 9 years in patients with hepatitis. The virus is transfusion-transmissible. It has a global distribution and is present within the volunteer blood donor population in the United States.
Asunto(s)
Virus de Hepatitis/genética , Hepatitis Viral Humana/virología , Virus ARN/genética , Reacción a la Transfusión , Enfermedad Aguda , Secuencia de Aminoácidos , Secuencia de Bases , Donantes de Sangre , Patógenos Transmitidos por la Sangre , Enfermedad Crónica , Clonación Molecular , Secuencia de Consenso , Transmisión de Enfermedad Infecciosa , Flaviviridae/genética , Genoma Viral , Virus de Hepatitis/química , Virus de Hepatitis/aislamiento & purificación , Hepatitis Viral Humana/epidemiología , Hepatitis Viral Humana/transmisión , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Virus ARN/química , Virus ARN/aislamiento & purificación , ARN Viral/sangre , ARN Viral/genética , Alineación de Secuencia , Estados Unidos/epidemiología , Proteínas Virales/química , Proteínas Virales/genética , Viremia/epidemiología , Viremia/virologíaRESUMEN
We have isolated and characterized the gene encoding mouse synexin, which consists of 14 exons and spans approximately 30 kbp of genomic DNA. The protein's unique N-terminal domain is encoded by six exons, and the C-terminal tetrad repeat, the site of the membrane-fusion and ion-channel domain, is encoded by seven exons. The first exon encodes the 5'-untranslated region. Analysis of synexin-gene expression in different mouse tissues shows that mRNA with exon 6 is only present in brain, heart and skeletal muscle. mRNA lacking exon 6 is expressed in all tissues we have examined. The initiation site for transcription was determined by primer-extension analysis and S1 nuclease mapping. Sequence analysis of the 1.3 kb 5'-flanking region revealed that the promoter has a TATA box located at position -25 and a number of potential promoter and regulatory elements. A CCAAT motif was not observed but CCATT is located in an appropriate position for the CCAAT motif upstream from the transcription-initiation start site. In addition, the 5'-flanking region contains two sets of palindromic sequences. Finally, we have determined that the functional synexin gene (Anx7) is located on mouse chromosome 14 and that a pseudogene (Anx7-ps1) is located on chromosome 10.
Asunto(s)
Anexina A7/genética , Mapeo Cromosómico , ADN/química , Células 3T3 , Secuencia de Aminoácidos , Animales , Anexina A7/química , Secuencia de Bases , Química Encefálica , Expresión Génica , Ratones , Datos de Secuencia Molecular , Músculos/química , Miocardio/química , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Mensajero/análisis , Mapeo Restrictivo , Análisis de Secuencia , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Distribución TisularRESUMEN
Two sets of cDNAs encoding mouse synexin were isolated from a liver cDNA library and sequenced. The coding regions of synexin clones show 99% identity. By contrast, the two mouse synexin cDNAs differ in a number of ways in both 5' and 3' non-coding regions. The two sets of cDNA encode a polypeptide of 463 amino acid residues which has a deduced molecular mass of 50 kDa. The amino acid sequence of mouse synexin shows a high degree of similarity to both the unique N-terminal domain and the highly conserved C-terminal domain of previously cloned human synexin. Northern-blot analysis using mouse liver polyadenylated RNA revealed two transcripts of 1.8 kb and 2.6 kb, corresponding to group I and group II respectively. Further hybridization analysis using specific sequences from each set of clones showed that the two sizes of mRNAs differ in the length of the 3' non-coding region which corresponded to the cDNAs. Both mouse liver synexin and recombinant mouse synexin expressed in Escherichia coli reacted after Western-blot analysis with a goat antibody against bovine synexin. Only in the larger group-II cDNAs do we find point mutations leading to amino acid replacements of Ser to Ala at residue 145 in the unique N-terminal domain, and of Ala to Gly at residue 304 in the transition zone between repeats II and III. We conclude from a comparison of mouse, human and Dictyostelium synexins that changes occur predominantly in the hydrophobic N-terminal domain, or, in the C-terminal region at the ends of some predicted alpha-helices, on the hydrophobic face of the amphipathic C-helices, and within a lengthy non-helical domain connecting major repeats II and III.
Asunto(s)
Anexina A7/genética , Ratones/genética , Filogenia , Polimorfismo Genético , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , Poli A/genética , Conformación Proteica , ARN Mensajero/genética , Homología de Secuencia de AminoácidoAsunto(s)
Canales de Calcio/fisiología , Fusión de Membrana/fisiología , Proteínas/fisiología , Animales , Anexina A7 , Fenómenos Biofísicos , Biofisica , Canales de Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/fisiología , Humanos , Modelos Moleculares , Familia de Multigenes , Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/fisiologíaRESUMEN
Our previous studies showed that the AP-1 recognition element (ARE) present within the SV40 72-base pair (bp) enhancer will activate transcription in yeast when placed upstream of a truncated CYC1 promoter. However, the AP-2/AP-3 recognition element (also known as the core sequence TGTGGAAAG) from the SV40 enhancer was not able to activate CYC1-dependent transcription. In this report, we show that the core sequence, when cloned next to a yeast UAS (upstream activation sequence), can inhibit the transcriptional stimulatory activity of the UAS. We refer to this sequence as the upstream repressor element (URE) in yeast. Repression occurs in an orientation-independent fashion and irrespective of the placement of the URE between the UAS and TATA box or upstream of both of these elements. Furthermore, repression is seen when the URE is separated from the UAS by up to 214 bp. Interestingly, multiple copies of an activator site can overcome this repression. Gel-shift analysis and URE-probed proteins blots indicate the presence of two polypeptide chains capable of binding the URE in yeast. The experimental evidence suggests that either the repression associated with the URE sequence is mediated by a direct, one-to-one interaction between the proteins recognizing the URE and GCRE, or alternatively, that there is a direct interaction between the activator and repressor for a general transcription factor.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Virus 40 de los Simios/genética , Secuencia de Bases , Clonación Molecular , Desoxirribonucleasa I , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plásmidos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Represoras/genética , Transcripción Genética , Activación Transcripcional , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
An assay that employs nucleoside 5'-O-(2-thiotriphosphates) was used to detect initiation of mouse mammary tumor virus (MMTV) RNA chains in preparations of isolated nuclei from cultured rat hepatoma cells containing stably integrated proviruses. RNA chains initiated with adenosine 5'-O-(2-thiotriphosphate), guanosine 5'-O-(2-thiotriphosphate), or uridine 5'-O-(2-thiotriphosphate) were separated from the remaining RNA by mercury-Sepharose column chromatography and analyzed for correctly initiated RNA chains with a T1 nuclease protection assay. Combined use of the thionucleotide transcription reaction with the T1 nuclease assay allowed precise localization of the transcription start sites. The majority of MMTV RNA chains were initiated with guanosine 5'-O-(2-thiotriphosphate) at a template site 133 nucleotides upstream from a PvuII site that coincides with the right end of the long terminal repeat. However, some RNA chains were also initiated with adenosine 5'-O-(2-thiotriphosphate) and uridine 5'-O-(2-thiotriphosphate) at template sites within three nucleotides of the primary guanosine start site. When Mn2+ was substituted for Mg2+ in the transcription reaction, MMTV RNA chains were initiated with approximately the same efficiency, but the start site was shifted to a position approximately 40 nucleotides downstream from the physiological start site; in the presence of Mn2+, MMTV RNA chains were initiated only with guanosine 5'-O-(2-thiotriphosphate). When the nuclei were exposed to both Mn2+ and Mg2+, transcription initiated at the manganese-dependent site. Mn2+ also caused the transcription start site for 45 S pre-rRNA to shift about 10 nucleotides upstream from the physiologically correct start site.
Asunto(s)
Cromatina/metabolismo , Magnesio/farmacología , Manganeso/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Cationes Bivalentes , Línea Celular Transformada , Núcleo Celular/metabolismo , Cromatina/efectos de los fármacos , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacología , Neoplasias Hepáticas Experimentales , Virus del Tumor Mamario del Ratón/genética , Hibridación de Ácido Nucleico , ARN Viral/genética , Moldes Genéticos , Tionucleótidos/farmacologíaRESUMEN
An assay that employs guanosine 5'-O-(2-thiotriphosphate) was used to measure correct initiation of RNA chains in isolated cell nuclei, where chromatin structure is relatively undisturbed. RNA chains initiated with guanosine 5'-O-(2-thiotriphosphate) were separated from the remaining RNA by mercury-Sepharose column chromatography and analyzed for correctly initiated mouse mammary tumor virus RNA with a T1 nuclease protection assay. The monovalent cation concentration dependence for initiation in isolated nuclei was similar to that previously observed for initiation from naked DNA templates (optimum near 90 mM) but different from that for elongation of nascent RNA chains. However, in contrast to the systems that employ naked DNA templates, initiation efficiency in the nuclear system was relatively unaffected by moderate changes in pH (6.7-8.3), temperature (25-37 degrees C), and magnesium ion concentration (1-9 mM). The optimized assay was used to assess the inhibitory activity of several compounds that have been reported to be specific inhibitors of transcription initiation on naked DNA templates. Both Sarkosyl and heparin were effective inhibitors of specific initiation by RNA polymerases I and II in isolated nuclei without inhibiting elongation of nascent chains, but 5,6-dichlorobenzimidazole riboside was relatively ineffective as a specific inhibitor of initiation by RNA polymerase II.