RESUMEN
HeLa cells are used to study the life cycles of many different viruses, including the human rhinoviruses (HRV) in the family Picornaviridae. Although the natural targets of HRV are human bronchial epithelial cells (hBE), it is generally more difficult to obtain and maintain the relevant primary cell cultures, relative to HeLa cells. Given that the HRV are now identified as a major cause of human asthma exacerbations, it becomes important to document how much of the virus biology learned from HeLa cells is common also to natural primary cells. When compared directly in matched infections using A01a virus, the kinetics of RNA replication, the synthesis and processing of viral proteins and the general subcellular localization of key non-structural proteins were resembled in hBE and HeLa cells. Viral-induced shutoff of host cell processes (e.g. nucleo-cytoplasmic trafficking) was also comparable.
Asunto(s)
Células Epiteliales/virología , Rhinovirus/fisiología , Células Cultivadas , Humanos , Rhinovirus/crecimiento & desarrollo , Cultivo de Virus , Replicación ViralRESUMEN
Interferons induced by viral infections can have powerful immuno- modulatory effects, and several epidemiologic studies have found an association between certain viral infections and reduced prevalence of allergy. We hypothesized that allergenic proteins could be synthesized by a replicating virus, and this construct could be useful as an immunomodulator. To test this hypothesis, we cloned an allergenic protein (ovomucoid [OVM]) into a murine picornavirus (Mengo virus) vector. This plasmid has a multicloning site surrounded by auto-catalytic sequences so that a foreign protein will be cleaved from viral proteins during replication. OVM sequences were cloned in the context of full-length viral genome cDNA, T7 RNA transcripts of this plasmid were transfected into HeLa cells, and recombinant virus plaques appeared on the second passage. Sequence analysis of recombinant viruses derived from individual plaques demonstrated that three viral isolates contained up to 2/3 of the OVM coding sequence, which was retained by the viruses after 5 additional passages in HeLa cells. The experiments verify the stable expression of immunoreactive OVM subunits by replicating viruses. These virus/allergen constructs could provide a tool to evaluate whether intracellular presentation of allergenic proteins in the context of a viral infection could prevent allergic sensitization upon re-challenge.
Asunto(s)
Alérgenos/biosíntesis , Mengovirus/fisiología , Ovomucina/biosíntesis , Virus Reordenados/fisiología , Alérgenos/genética , Animales , Infecciones por Cardiovirus/virología , Vectores Genéticos/metabolismo , Vectores Genéticos/fisiología , Genoma Viral/genética , Células HeLa/metabolismo , Humanos , Mengovirus/genética , Mengovirus/metabolismo , Ratones , Ovomucina/genética , Ovomucina/inmunología , Plásmidos/genética , Virus Reordenados/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Recombinación Genética , Pase Seriado , Especificidad de la Especie , Transfección , Replicación ViralRESUMEN
Human rhinovirus (HRV) 3C protease (3Cpro) plays several important roles in the virus replication cycle. This enzyme cleaves the viral polyprotein at discrete sites to produce mature viral proteins and also inhibits cellular RNA transcription. It is not clear, however, whether the observed transcriptional shutoff activities are due to 3Cpro itself or to 3Cpro-containing precursors, and where 3Cpro exerts its effects within infected cells. To address these questions HeLa cells were infected with HRV-16, stained with polyclonal antibodies directed against 3Cpro and then analysed by laser confocal microscopy. Proteins containing 3Cpro accumulated in nuclei 2-4 h post-infection, and progressively increased in the cytoplasm. Analyses of subcellular extracts demonstrated that 3CD', a minor component among 3Cpro precursors, gave rise to the earliest 3Cpro nuclear signals. Mature 3Cpro and another 3Cpro precursor, 3CD, were also detected in the nucleus, cytoplasm and perinuclear membrane fractions 4 h post-infection. Transfecting cells with 3Cpro, 3CD precursor and 3CD(Delta371) (with deletion of 371 aa at the carboxyl terminus of 3D) demonstrated that the nucleolar localization signal was near the amino terminus of 3D. In addition, 3Cpro precursors were found to co-localize in nuclei with the transcription factor OCT-1 and the nucleolar chaperone B23. Finally, it was demonstrated that HRV-16 3Cpro, 3CD and 3CD(Delta371) could cleave OCT-1. Collectively, these findings suggest that HRV 3CD' and/or 3CD are specifically localized to the nucleoli of infected cells during the early stage of infection, and contribute to the inhibition of cellular RNA transcription via a proteolytic mechanism.
Asunto(s)
Transporte Activo de Núcleo Celular , Núcleo Celular/enzimología , Cisteína Endopeptidasas/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas Virales/metabolismo , Proteasas Virales 3C , Secuencia de Aminoácidos , Fraccionamiento Celular , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Factor 1 de Transcripción de Unión a Octámeros , Factores de Transcripción/metabolismoRESUMEN
Synthesis, cDNA cloning, and nucleotide sequencing of F gene of rinderpest virus strain K was carried out. Analysis of nucleotide sequence showed the only open reading frame coding for protein from 546 a.o. with mol. weight 58.6 kDa. The mean percentage of identical nucleotide residues between F genes of strains K, Kabete O, and L is 76.4% for 5'-untranslated region and 90.5% for translated region, the share of similar amino acid residues in the respective proteins is 92.9%. The structure of restriction site of F0 precursor protein in rinderpest strains with different virulence is similar. Protein F of rinderpest virus strain K has 3 potential glycosylation sites and 13 cystein residues in positions identical to those of F protein of rinderpest strains Kabete O and L.
Asunto(s)
Genes Virales , Glicoproteínas/genética , Virus de la Peste Bovina/genética , Proteínas Virales de Fusión/genética , Regiones no Traducidas 5' , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , ADN Complementario , Glicoproteínas/química , Proteínas de la Membrana , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteínas Virales de Fusión/químicaRESUMEN
Auesku disease virus--suid herpesvirus type I (SHV-1) genome is analyzed by the restriction fragment pattern analysis. The analyzed strains isolated in CIS countries in 1962-1992 were compared with the European strains. Genomes of field strains Arsk, Turkmenia, and Geneva differed and even lacked the restriction fragments typical of vaccine strains MK-25 or UNIIEV-18S, which agrees with the results of the neutralization test. Immunobiological characteristics of SHV-1 strains are discussed.
Asunto(s)
Genoma Viral , Herpesvirus Suido 1/genética , Seudorrabia/virología , Animales , Comunidad de Estados Independientes/epidemiología , Europa (Continente)/epidemiología , Herpesvirus Suido 1/aislamiento & purificación , Seudorrabia/epidemiología , Mapeo Restrictivo , Análisis de Secuencia de ADNRESUMEN
Synthesis, cDNA cloning, and identification of H gene nucleotide sequence of rinderpest virus (RPV) K strain are carried out. Analysis of the identified nucleotide sequence has revealed the single open reading frame encoding a protein consisting of 609 amino acids with molecular weight of 68 kDa. The mean nucleotide homology between H genes of K, Kabete O and L strains in 88.0%, the mean amino acid homology of the corresponding proteins is 88.2%. RPV K strain hemagglutinin contains 5 potential glycosylation sites. The position of all 13 cystein bases is identical to positions in H proteins of RPV Kabete O and L strains. Studies of the hydrophobic profile of the compared proteins have shown 2 potential transmembrane fragments.
Asunto(s)
ADN Viral/genética , Virus de la Peste Bovina/genética , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , ADN Complementario , Genes Virales , Datos de Secuencia Molecular , Proteínas Estructurales Virales/genéticaAsunto(s)
Genoma Viral , Herpesvirus Gallináceo 2/genética , Reacción en Cadena de la Polimerasa/métodos , Antígenos Virales/genética , Secuencia de Bases , Cartilla de ADN , Sondas de ADN , ADN Viral , Genes Virales , Herpesvirus Gallináceo 2/clasificación , Herpesvirus Gallináceo 2/enzimología , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Timidina Quinasa/genética , Proteínas del Envoltorio Viral/genéticaAsunto(s)
Aphthovirus/enzimología , Cisteína Endopeptidasas/genética , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Fiebre Aftosa/diagnóstico , Proteínas Recombinantes de Fusión , Proteínas Virales , Proteasas Virales 3C , Animales , Aphthovirus/inmunología , Técnicas para Inmunoenzimas , Conejos , Proteínas Recombinantes de Fusión/genética , Porcinos , Vacunas Sintéticas , Vacunas ViralesRESUMEN
Cells of a suspension HeLa culture at the logarithmic phase of growth were exposed to 60Co-gamma-rays (5 Gy), incubated in the nutritious medium, and in 4 h subjected to repeated irradiation: the dose-response function and the dynamics of DNA synthesis inhibition were determined. It was shown that DNA synthesis was inhibited to a lesser extent after preirradiation, in other words, DNA synthesis was radioresistant. A correlation between this synthesis and reproductive cell death is discussed.
Asunto(s)
Daño del ADN , Replicación del ADN/efectos de la radiación , ADN/efectos de la radiación , Tolerancia a Radiación , ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Floxuridina/farmacología , Células HeLa , HumanosRESUMEN
A gamma-radiation dose (Di) suppressing DNA synthesis initiation by 35% in primary suspension cultures of mammalian cells, is nearly the same as D0 for survival of clonogenic cells of the same lines and tissues. The extent of DNA synthesis suppression is assessed by impulse 3H-thymidine incorporation in the acid-insoluble fraction of irradiated cells. The values of Di determined in this way for HeLa cells, Ehrlich ascites tumor cells, mouse bone marrow and thymus cells are 2.0, 1.5, 1.5, and 1.0 Gy, respectively; as determined by clonogenic capacity of these cells, Di = 1.9, 2.0, 1.3, and 1.0 Gy, respectively.