RESUMEN
We compared the effects of polypyrimidine tract-binding protein (PTB) on hepatitis C virus (HCV genotype IIa), encephalomyocarditis virus (EMCV) and poliovirus internal ribosome entry site (IRES) activities in vitro. It bound strongly to EMCV IRES, but weakly to PV and HCV RNAs. PV IRES showed the strongest dependency to PTB and it showed less than one-tenth of IRES activity after the immuno-depletion of PTB from HeLa S10 lysate with pre-coated anti-PTB IgG beads, comparing to the normal IgG beads-treated S10 lysate. EMCV IRES activity was approximately 40% of that of normal control after PTB depletion. Especially, HCV IRES activity was approximately 95%, and most weekly affected by the depletion of PTB. Repletion of PTB to depleted S10 lysate restored activities of PV and EMCV IRESs. The data suggest that PTB plays an important role in picornaviral IRESs, but not in HCV IRES.
Asunto(s)
Virus de la Encefalomiocarditis/genética , Regulación Viral de la Expresión Génica/fisiología , Hepacivirus/genética , Poliovirus/genética , Proteína de Unión al Tracto de Polipirimidina/fisiología , Sitio de Iniciación de la Transcripción/fisiología , Animales , Anticuerpos Antivirales/biosíntesis , Cobayas , Células HeLa , Humanos , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Biosíntesis de Proteínas , ARN Viral/metabolismo , ConejosRESUMEN
Hepatitis C virus (HCV) causes persistent infection in most patients. To clarify the mechanisms underlying establishment of this persistent infection, nucleotide sequences of the E1/E2 region were characterized in 5 patients with acute and chronic HCV infection. We used direct DNA sequencing methods to identify the major sequence of HCV in each patient. Each HCV genome displayed a high frequency of nucleotide sequence variation in the hypervariable region (HVR) of E2. However, patient-specific conserved nucleotide sequences were identified in the E1/E2 region during the course of infection and conserved the higher-order protein structure. In the acute phase HCV infection, amino acid substitution in HVR-1 as the monthly rate of amino acids substitution per site (%) between each point exceeded 10.2%. In the chronic phase HCV infection, a significantly lower rate of amino acid substitution was observed in patients. The host immune responses to HVR-1 of each HCV isolates from all clinical courses were characterized using synthetic peptides and ELISA. One chronic patient serum (genotype 1b) did not react at all to its own HVR-1 peptides, however another patient (genotype 2b) reacted to all clinical course. These results indicated that HVR-1 might not always exhibit neutralizing epitopes of HCV infection. The sequence variation in HVR-1 may instead indicate the existence of various clones in acute phase infection and the adaption of these clones is thought to have caused persistent and chronic infection in each patient.
Asunto(s)
Hepacivirus/clasificación , Hepacivirus/patogenicidad , Análisis de Secuencia de ADN , Proteínas del Envoltorio Viral/química , Proteínas Virales/química , Enfermedad Aguda , Adulto , Anciano , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Enfermedad Crónica , Femenino , Variación Genética , Genotipo , Hepacivirus/genética , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas del Envoltorio Viral/genética , Proteínas Virales/genéticaRESUMEN
The swine interleukin-4 (SwIL-4) cDNA was cloned by RT-PCR. It was expressed using an expression vector pQE30 in E. coli, a baculovirus AcNPV vector pVL1392 in insect cells, and a pCAGGS vector in mammalian cells. The rSwIL-4 proteins expressed from bacteria and insect cells were purified using a chelating affinity column and a mAb-coupled immunoaffinity column. The amount of the products and their bioactivities were compared. All recombinant cytokines were efficiently reacted with the specific antibodies and the molecular weight of rSwIL-4 was approximately 16 kDa in E. coli, 15 and 18 kDa in insect cells, and 15 and 20 kDa in mammalian cells. Variations of molecular weight observed in insect and mammalian cells were probably due to different modification ways of glycosylation. All these recombinant proteins retained their antigenicity and were biologically active in inducing human TF-1 cell proliferation in vitro. The simple purification method will make it possible to evaluate the in vitro and in vivo effects of IL-4 in pigs.
Asunto(s)
Interleucina-4/biosíntesis , Proteínas Recombinantes/biosíntesis , Animales , Baculoviridae , Línea Celular , Proliferación Celular/efectos de los fármacos , Clonación Molecular , Escherichia coli , Vectores Genéticos , Humanos , Interleucina-4/aislamiento & purificación , Interleucina-4/farmacología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , PorcinosRESUMEN
The swine interleukin-6 (SwIL-6) cDNA was cloned by RT-PCR and each expression system of recombinant SwIL-6 in Escherichia coli, insect cells, and mammalian cells was developed. Recombinant SwIL-6 produced in bacteria was applied for generation of the polyclonal antibodies. The rSwIL-6 was purified from supernatant of insect cells with a Q-sepharose or anti-SwIL-6 monoclonal antibody based immunoaffinity column. The antibodies showed that the molecular weight of rSwIL-6 was approximately 26kDa in E. coli, 25, 26, 30kDa in insect cells, and 26 and 30kDa in mammalian cells. These variations of molecular weight were probably due to the different modifications of glycosylation. All these recombinant proteins retained the antigenicity and biological activity on 7TD1 mouse cells.
Asunto(s)
Interleucina-6/biosíntesis , Interleucina-6/genética , Porcinos/genética , Animales , Anticuerpos Monoclonales/inmunología , Formación de Anticuerpos/efectos de los fármacos , Baculoviridae/genética , Bioensayo/veterinaria , Western Blotting/veterinaria , Células COS , Chlorocebus aethiops , Clonación Molecular , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida/veterinaria , Escherichia coli/genética , Escherichia coli/metabolismo , Interleucina-6/inmunología , Interleucina-6/aislamiento & purificación , Ratones , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Análisis de Secuencia de ADN , Spodoptera/metabolismo , Spodoptera/virología , Porcinos/inmunología , TransfecciónRESUMEN
We established a sandwich enzyme-linked immunosorbent assay (ELISA) for swine interleukin-6 (SwIL-6), which was applied for detection of SwIL-6 in vitro and in vivo. Anti-SwIL-6 rabbit- and goat-polyclonal antibodies, and monoclonal antibody (mAb) were prepared, conforming that all of the antibodies were reactive with recombinant SwIL-6 by Western blotting and indirect ELISA. A sandwich ELISA was developed using the mAb as a capture antibody and biotinylated goat-polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA for rSwIL-6 was 49pg/ml and did not show cross-reactivity with swine IL-1b, IL-4, IL-8, IL-18, IL-12, and IFN-g. Using the ELISA, SwIL-6 was detected in culture medium of the monocytes stimulated with PHA-P and PMA, and the plasma or the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Actinobacillus pleuropneumoniae or Mycoplasma hyopneumoniae. This ELISA for SwIL-6 may be useful for understanding the role of this cytokine in various swine diseases.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/veterinaria , Interleucina-6/análisis , Porcinos/inmunología , Infecciones por Actinobacillus/inmunología , Infecciones por Actinobacillus/veterinaria , Actinobacillus pleuropneumoniae/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting/veterinaria , Líquido del Lavado Bronquioalveolar/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Mycoplasma hyopneumoniae/inmunología , Neumonía Porcina por Mycoplasma/inmunologíaRESUMEN
The in vitro effect and the in vivo influence of recombinant swine IL-4 (rSwIL-4) were characterized in various swine cells and in nursery pigs on LPS-induced endotoxic shock and pro-inflammatory cytokine productions. In in vitro experiment, the rSwIL-4 induced a proliferation of CD4 positive T cells in mitogen-prestimulated peripheral blood mononuclear cell (PBMC). In addition, the rSwIL-4, which was produced from insect cells, promoted the differentiation of monocytes into immature dendritic cells in combination with granulocyte macrophage-colony stimulating factor (GM-CSF). Furthermore, the rSwIL-4 successfully suppressed the LPS-induced secretion of TNF-alpha, IL-1alpha, IL-6, IL-8, and IL-18 from swine alveolar macrophages when rSwIL-4 was treated at the same time with LPS. In in vivo experiment in nursery pigs, subcutaneous pretreatment of rSwIL-4, which was produced from baculovirus expression system, enhanced the severity of respiratory failure with endotoxic shock, and increased the production of TNF-alpha and IL-18 in response to inoculation with LPS. These results indicate that the rSwIL-4 is biologically active in both in vitro and in vivo treatments. Depending on the administration time, pro-inflammatory cytokine productions by IL-4 can cause either inhibitory or stimulatory regulation.
Asunto(s)
Interleucina-4/farmacología , Monocitos/efectos de los fármacos , Enfermedades Respiratorias/veterinaria , Choque Séptico/veterinaria , Enfermedades de los Porcinos/inmunología , Porcinos/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Células COS , Chlorocebus aethiops , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Citometría de Flujo/veterinaria , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Interleucina-18/inmunología , Interleucina-4/inmunología , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/inmunología , Monocitos/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Enfermedades Respiratorias/tratamiento farmacológico , Enfermedades Respiratorias/inmunología , Choque Séptico/tratamiento farmacológico , Choque Séptico/inmunología , Organismos Libres de Patógenos Específicos , Enfermedades de los Porcinos/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The Yanaka strain, a field isolate of Canine distemper virus (CDV), caused extensive syncytial cytopathic effects (CPEs) followed by cell death in vitro. Syncytium formation is an important aspect of CDV pathogenicity, but the mechanism of the fusion-induced cell death is still not understood. In this study, the involvement of apoptosis in the CDV-induced CPE was investigated. We also examined apoptosis in cells infected with a persistent strain of CDV, the Yanaka-BP strain derived from the Yanaka strain, because this strain does not cause obvious CPE. DNA laddering together with Terminal transferase dUTP nick endlabeling (TUNEL) assay indicated that the Yanaka strain infection, but not the Yanaka-BP infection induced apoptosis. In addition, flow cytometric analysis similarly indicated that the Yanaka-BP strain induced apoptosis significantly less frequently than the Yanaka strain did. Thus, absence of apoptosis may be implicated in the CPE and establishment of persistent CDV infection.
Asunto(s)
Apoptosis , Virus del Moquillo Canino/patogenicidad , Células Gigantes/patología , Animales , Anexina A5/metabolismo , Línea Celular , Efecto Citopatogénico Viral , Fragmentación del ADN , Virus del Moquillo Canino/clasificación , Perros , Citometría de Flujo , Células Gigantes/metabolismo , Etiquetado Corte-Fin in Situ , Especificidad de la EspecieRESUMEN
We produced four monoclonal antibodies (mAb) and two polyclonal antibodies using the purified cytokine expressed in bacteria and characterized them. Specific binding of each of the mAb and polyclonal antibodies to recombinant swine IL-4 (rSwIL-4) purified from Escherichia coli and baculovirus was demonstrated in an indirect ELISA and/or in western blotting. We established a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring concentration of SwIL-4 in biological samples and established an enzyme-linked immunospot (ELISPOT) assay for detecting IL-4-secreting cells using a mAb and a polyclonal IgG from goat. The detection limit of the sandwich ELISA for SwIL-4 was 78 pg/ml. Using sandwich ELISA, SwIL-4 was detected in the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Mycoplasma hyopneumoniae and could quantitate in supernatants of mitogen-stimulated PBMC culture. The ELISPOT system is useful for the detection of IL-4 producing cells in swine PBMC culture.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Interleucina-4/inmunología , Porcinos/inmunología , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Líquido del Lavado Bronquioalveolar/inmunología , Células Cultivadas , Cabras , Interleucina-4/biosíntesis , Ratones , Infecciones por Mycoplasma/inmunología , Infecciones por Mycoplasma/veterinaria , Mycoplasma hyopneumoniae , Conejos , Enfermedades de los Porcinos/inmunología , Células Th2/inmunologíaRESUMEN
All nuclear-encoded mRNAs contain a 5' cap structure (m7GpppN, where N is any nucleotide), which is recognized by the eukaryotic translation initiation factor 4E (eIF4E) subunit of the eIF4F complex. The eIF4E-binding proteins constitute a family of three polypeptides that reversibly repress cap-dependent translation by binding to eIF4E, thus preventing the formation of the eIF4F complex. We investigated the biological function of 4E-BP1 by disrupting its gene (Eif4ebp1) in the mouse. Eif4ebp1-/- mice manifest markedly smaller white fat pads than wild-type animals, and knockout males display an increase in metabolic rate. The males' white adipose tissue contains cells that exhibit the distinctive multilocular appearance of brown adipocytes, and expresses the uncoupling protein 1 (UCP1), a specific marker of brown fat. Consistent with these observations, translation of the peroxisome proliferator-activated receptor-gamma co-activator 1 (PGC1), a transcriptional co-activator implicated in mitochondrial biogenesis and adaptive thermogenesis, is increased in white adipose tissue of Eif4ebp1-/- mice. These findings demonstrate that 4E-BP1 is a novel regulator of adipogenesis and metabolism in mammals.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas de la Membrana/genética , Factores de Iniciación de Péptidos/fisiología , Fosfoproteínas/fisiología , Biosíntesis de Proteínas , Proteínas Represoras/fisiología , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales , Tejido Adiposo/metabolismo , Animales , Metabolismo Basal , Proteínas de Ciclo Celular , Factor 4E Eucariótico de Iniciación , Factores Eucarióticos de Iniciación , Regulación de la Expresión Génica , Marcación de Gen , Hipoglucemia/metabolismo , Canales Iónicos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas Mitocondriales , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , Fosfoproteínas/genética , Fosforilación , ARN Mensajero , Proteínas Represoras/genética , Proteína Desacopladora 1RESUMEN
Persistent hepatitis C virus (HCV) infection often progresses to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Numerous viruses have been reported to escape from apoptotic mechanism to maintain persistent infection. In the present study, we characterized the effect of HCV proteins on the Fas signal using HCV transgenic mice, which expressed core, E1, E2, and NS2 proteins, regulated by the Cre/loxP switching system. The transgene expression of HCV transgenic mice caused resistance to Fas antibody stimulated lethality. Apoptotic cell death in the liver of HCV protein expressing mice was significantly reduced compared with nonexpressing mice. Histopathological analysis and DNA fragmentation analysis revealed that the HCV proteins suppressed Fas-mediated apoptotic cell death. To identify the target pathway of HCV proteins, we characterized caspase activity. The activation of caspase-9 and -3/7 but not caspase-8 was inhibited by HCV proteins. Cytochrome c release from mitochondria was inhibited in HCV protein expressing mice. These results indicated that the expression of HCV proteins may directly or indirectly inhibit Fas-mediated apoptosis and death in mice by repressing the release of cytochrome c from mitochondria, thereby suppressing caspase-9 and -3/7 activation. These results suggest that HCV may cause persistent infection, as a result of suppression of Fas-mediated cell death.
Asunto(s)
Grupo Citocromo c/metabolismo , Hepacivirus/metabolismo , Transducción de Señal/fisiología , Proteínas Virales/metabolismo , Receptor fas/fisiología , Animales , Apoptosis/fisiología , Caspasas/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones TransgénicosRESUMEN
We previously showed that two proteins with molecular weights of 56 and 58 kDa are produced from nonstructural protein 5A (NS5A) derived from hepatitis C virus (HCV)-1b genotype. The 56-kDa protein is phosphorylated at serine residues in NS5A, including those located in the C-terminal region of NS5A, while the 58-kDa protein, the hyperphosphorylated form of the 56-kDa protein, is phosphorylated at serine residues in the central region. This hyperphosphorylation is dependent on the presence of HCV NS4A protein. To clarify whether NS4A-dependent phosphorylation also occurs in other HCV genotypes, phosphorylation of NS5A was analyzed by two-dimensional gel electrophoresis. Here, we report that NS5A from the HCV-2a genotype was phosphorylated. However, hyperphosphorylation of NS5A occurs in the HCV-1b genotype but not in the -2a genotype. This result suggests that modification of NS5A phosphorylation reflects the virological features of HCV and that there are physiological differences in the roles of differently phosphorylated NS5A between HCV genotypes.
Asunto(s)
Hepacivirus/metabolismo , Proteínas no Estructurales Virales/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Células COS , Electroforesis en Gel Bidimensional , Fosforilación , TransfecciónRESUMEN
BACKGROUND & AIMS: For diagnosis of hepatitis C virus infection and monitoring of viral load in patients, a highly sensitive and accurate hepatitis C virus quantification system is essential. METHODS: Hepatitis C virus genome was detected by real/time detection system using an ABI Prism 7700 sequence detector (Perkin Elmer Corp./Applied Biosystems, Foster City, CA). RESULTS: As few as 10 copies of the genome were detected, and the quantification range was between 10(1) and 10(8) copies (r > 0.99). This system was 10-100-fold more sensitive than an Amplicor monitor (Roche Diagnostic Systems, Branchburg, NJ). The coefficient of variation values for both intra-assay precision and interassay reproducibility of identifying the genome quantification ranged from 0.37% to 2.00% and 0.88% to 4.66%, respectively. The system could detect the genome in 98% of patients with chronic hepatitis, 95.8% of patients with liver cirrhosis, and 100% of patients with hepatocellular carcinoma who had the antibody to hepatitis C virus, but could not detect the genome in patients without the antibody. CONCLUSIONS: The establishment of a real-time detection system enables more accurate diagnosis of infection and monitoring of viral load in interferon-treated patients via quantification of viral genome.
Asunto(s)
Genoma Viral , Hepacivirus/genética , Hepatitis C/diagnóstico , Reacción en Cadena de la Polimerasa/instrumentación , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/sangre , Carcinoma Hepatocelular/virología , Genotipo , Hepacivirus/aislamiento & purificación , Anticuerpos contra la Hepatitis C/sangre , Humanos , Cirrosis Hepática/virología , Neoplasias Hepáticas/virología , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Transcripción GenéticaRESUMEN
The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles.
Asunto(s)
Hepacivirus/química , Proteínas del Núcleo Viral/fisiología , Animales , Western Blotting , Centrifugación por Gradiente de Densidad , Microscopía Confocal , Conformación Proteica , ARN Viral/análisis , Conejos , Proteínas del Núcleo Viral/análisis , Proteínas del Núcleo Viral/químicaRESUMEN
Polypyrimidine-tract-binding protein (PTB) is involved in pre-mRNA splicing and internal-ribosomal-entry-site-dependent translation. The biochemical properties of various segments of PTB were analysed in order to understand the molecular basis of the PTB functions. The protein exists in oligomeric as well as monomeric form. The central part of PTB (amino acids 169-293) plays a major role in the oligomerization. PTB contains several RNA-binding motifs. Among them, the C-terminal part of PTB (amino acids 329-530) exhibited the strongest RNA-binding activity. The N-terminal part of PTB is responsible for the enhancement of RNA binding by HeLa cell cytoplasmic factor(s).
Asunto(s)
Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Reactivos de Enlaces Cruzados , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Proteína de Unión al Tracto de Polipirimidina , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/genética , Análisis de Secuencia , Relación Estructura-ActividadRESUMEN
Hepatitis C virus (HCV) carries an internal ribosomal entry site (IRES) within the 5' portion of the RNA. To identify structures that influence efficiency of the translation initiation, relative activities of modified IRESs were examined by using engineered bicistronic mRNAs, between the two cistrons of which various mutant IRESs were inserted. An IRES derived from genotype 2b is at least two times more efficient than one from genotype 1b in cultured cells. Activity ratios of genotype 2b IRES to 1b IRES differ in magnification among cultured cells, suggesting the difference in assortment of IRES-related host factors among individual cell types. Recombinant IRESs between the genotypes show similar or higher activities compared with 2b IRES in cell-free systems and show intermediate activities in cultured cells. Patterns of relative activities of those IRESs indicate that the IRES activity is not regulated by defined structure(s), although a cluster of different nucleotides is observed in the genome region of nucleotides 176-224 between the two alleles. The results suggest that a highly ordered structure formed by the entire 5' portion of the RNA is important for the IRES activity. The 5' border of HCV IRES was examined by using a series of deletion RNAs in various systems. The results strongly suggest that the border resides between nucleotide positions 28 and 45. Patterns of relative activities of the deletion IRESs differ in translation systems or cell types. These results imply that interactions of HCV RNA with the related transacting factor(s) may differ in the translation systems or cell types.
Asunto(s)
Hepacivirus/genética , ARN Viral , Secuencia de Bases , Sitios de Unión , Sistema Libre de Células , Mapeo Cromosómico , Genotipo , Células HeLa , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Recombinación Genética , Ribosomas , Relación Estructura-ActividadRESUMEN
Two related eukaryotic initiation factor-4E binding proteins (4E-BP1 and 4E-BP2) were recently characterized for their capacity to bind specifically to eIF4E and inhibit its function. Here, we determined the cDNA sequence, tissue distribution, genomic structure, and chromosome localization of murine and human 4E-BP1 and 4E-BP2. Mouse 4E-BP1 and 4E-BP2 consist of 117 and 120 amino acids and exhibit 91. 5 and 95.0% identity, respectively, to their human homologues. 4E-BP1 mRNA is expressed in most tissues, but is most abundant in adipose tissue, pancreas, and skeletal muscle, while 4E-BP2 mRNA is ubiquitously expressed. The structures of the mouse 4E-BP1 and 4E-BP2 were determined. The 4E-BP1 gene consists of three exons and spans approximately 16 kb. In addition, two 4E-BP1 pseudogenes exist in the mouse genome. The 4E-BP2 gene spans approximately 20 kb and exhibits an identical genomic organization to that of 4E-BP1, with the protein coding portion of the gene divided into three exons. There are no pseudogenes for 4E-BP2. The chromosomal locations of 4E-BP1 and 4E-BP2 were determined in both mice and humans by fluorescence in situ hybridization analysis. Mouse 4E-BP1 and 4E-BP2 map to chromosomes 8 (A4-B1) and 10 (B4-B5), respectively, and human 4E-BP1 and 4E-BP2 localize to chromosomes 8p12 and 10q21-q22, respectively.
Asunto(s)
Proteínas Portadoras/genética , Factores Eucarióticos de Iniciación , Genes , Ratones/genética , Factores de Iniciación de Péptidos/metabolismo , Fosfoproteínas/genética , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular , Mapeo Cromosómico , Cromosomas Humanos Par 10/genética , Cromosomas Humanos Par 8/genética , Cruzamientos Genéticos , Factor 4E Eucariótico de Iniciación , Humanos , Hibridación Fluorescente in Situ , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Muridae/genética , Especificidad de Órganos , Seudogenes , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Responses of patients infected with chronic hepatitis C virus (HCV) to interferon-alpha (IFN-alpha) treatment were studied. HCV genotypes were determined by molecular and serologic techniques. Levels of HCV viremia were determined by quantitative reverse transcription-polymerase chain reaction. Infection with HCV genotype 2 or low pretreatment HCV viremia levels in subjects infected with genotype 1 were associated with favorable (complete and sustained) responses (CR-SR; P < .001) to IFN-alpha treatment. HCV viremia levels in genotype 2 infection were significantly lower (P < .05) than in genotype 1 infection. The reduction rates of serum HCV RNA levels were four times higher in patients with genotype 2 infection than in those infected with genotype 1. The proportion of patients with CR-SR who experienced virologic relapse after completion of IFN-alpha treatment was higher for those infected with genotype 1. The proportion of patients with CR-SR and genotype 1 infection increased linearly in accordance with increases in single or total IFN-alpha dose.
Asunto(s)
Antivirales/uso terapéutico , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Adulto , Anciano , Alanina Transaminasa/sangre , Relación Dosis-Respuesta a Droga , Femenino , Genotipo , Hepacivirus/clasificación , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Viral/sangre , Serotipificación , Especificidad de la EspecieAsunto(s)
Hepacivirus/genética , Hepatitis C/virología , Mutagénesis , Animales , Enfermedad Crónica , Genotipo , HumanosRESUMEN
In vitro translation experiments involving reticulocyte and HeLa cell lysates have demonstrated that translation initiation on hepatitis C virus (HCV) RNA occurs by entry of ribosomes to the internal sequence (internal ribosomal entry site [IRES]) within the 5' noncoding region (5'NCR). Specific binding factors that bind to the HCV IRES were examined by UV cross-linking tests. A protein p57 (polypyrimidine tract binding protein [PTB]), known to be a binding factor that binds to picornavirus IRES, was found to bind to the HCV IRES. To investigate functions of this PTB in IRES-dependent translation initiation, hyperimmune antibodies to PTB (anti-PTB) were prepared by immunizing rabbits with purified recombinant PTB produced in E. coli. Anti-PTB IgGs were purified and used to deplete PTB in a cell-free protein synthesis system prepared from HeLa cells. In vitro translation experiments using PTB-depleted lysates suggested that host factor dependency of IRES is different in individual IRESs. As we showed previously, function of the IRES typical of group II HCV is more efficient than the IRES typical of group I HCV in a cell-free protein synthesis system from HeLa cells. Recombinant HCV IRESs were constructed and tested for their function in the in vitro system. Most recombinant IRESs showed an initiation activity of the more efficient typical group II IRES level or higher. This observation, together with the fact that group I HCV is the most common isolate of HCV worldwide, suggests that the efficiency of the IRES function of HCV may be generally suppressed in nature, and that the replication ability of HCV that permits its persistent infection in humans may require a delicate balance at a molecular level.