RESUMEN
Negri bodies (NBs) are formed in the cytoplasm of rabies virus (RABV)-infected cells and are accompanied by a number of host factors to NBs, in which replication and transcription occur. Here, it was found that chaperonin containing TCP-1 subunit alpha (CCTα) relocalizes to NBs in RABV-infected cells, and that cotransfection of nucleo- and phospho-proteins of RABV is sufficient to recruit CCTα to the NBs' structure. Inhibition of CCTα expression by specific short hairpin RNA knockdown inhibited the replication and transcription of RABV. Therefore, this study showed that the host factor CCTα is associated with RABV infection and is very likely required for efficient virus transcription and replication.
Asunto(s)
Chaperonina con TCP-1/metabolismo , Virus de la Rabia/genética , Rabia/enzimología , Rabia/virología , Transcripción Genética , Replicación Viral , Animales , Línea Celular , Chaperonina con TCP-1/genética , Interacciones Huésped-Patógeno , Humanos , Cuerpos de Inclusión Viral/enzimología , Cuerpos de Inclusión Viral/virología , Ratones , Transporte de Proteínas , Rabia/genética , Virus de la Rabia/fisiologíaRESUMEN
The effects of the adenovirus infection on the distribution of the cellular protein kinase CK2 and double-stranded RNA-activated protein kinase (PKR) were examined at the ultrastructural level. Immunogold labeling revealed the redistribution of CK2 subunits and PKR to morphologically distinct structures of the cell nucleus. The electron-clear amorphous structures, designated pIX nuclear bodies in our previous work (Rosa-Calatrava et al., 2001), contained CK2 alpha and PKR. The protein crystals, which result from the regular assembly of hexon, penton base, and fiber proteins [Boulanger et al. (1970) J Gen Virol 6:329-332], contained CK2 beta and PKR. Both viral structures were devoid of viral RNA, including the PKR-inhibitor VA1 RNA generated by the RNA polymerase III. Instead, VA1 RNA accumulated in PKR-free viral compact rings in which the viral RNA generated by the RNA polymerase II was excluded.
Asunto(s)
Adenovirus Humanos/patogenicidad , Núcleo Celular/ultraestructura , Cuerpos de Inclusión Viral/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , eIF-2 Quinasa/metabolismo , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Quinasa de la Caseína II , Células HeLa/ultraestructura , Células HeLa/virología , Humanos , Hibridación in Situ , Microscopía Electrónica , ARN Bicatenario/metabolismo , ARN Ribosómico/metabolismo , ARN Viral/metabolismoRESUMEN
The gene encoding the C-terminal protease domain of the nuclear inclusion protein a (NIa) of tobacco vein mottling virus (TVMV) was cloned from an isolated virus particle and expressed as a fusion protein with glutathione S-transferase in Escherichia coli XL1-blue. The 27-kDa protease was purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography. The purified protease exhibited the specific proteolytic activity towards the nonapeptide substrates, Ac-Glu-Asn-Asn-Val-Arg-Phe-Gln-Ser-Leu-amide and Ac-Arg-Glu-Thr-Val-Arg-Phe-Gln-Ser-Asp-amide, containing the junction sequences between P3 protein and cylindrical inclusion protein and between nuclear inclusion protein b and capsid protein, respectively. The Km and k(cat) values were about 0.2 mM and 0.071 s(-1), respectively, which were approximately five-fold lower than those obtained for the NIa protease of turnip mosaic potyvirus (TuMV), suggesting that the TVMV NIa protease is different in the binding affinity as well as in the catalytic power from the TuMV NIa protease. In contrast to the NIa proteases from TuMV and tobacco etch virus, the TVMV NIa protease was not autocatalytically cleaved into smaller proteins, indicating that the C-terminal truncation is not a common phenomenon occurring in all potyviral NIa proteases. These results suggest that the TVMV NIa protease has a unique biochemical property distinct from those of other potyviral proteases.
Asunto(s)
Endopeptidasas/genética , Endopeptidasas/aislamiento & purificación , Potyviridae/genética , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/química , Endopeptidasas/metabolismo , Fluorescamina/química , Genes Reporteros , Cuerpos de Inclusión Viral/enzimología , Cuerpos de Inclusión Viral/genética , Datos de Secuencia Molecular , Plantas Tóxicas , Potyviridae/enzimología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Nicotiana/virología , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Cells of healthy maize plants as well as those infected by maize dwarf mosaic potyvirus were examined by electron microscopy for the location of ATPase activity. In healthy and virus infected plants, ATPase activity was found in plasma membranes, chloroplast thylakoid membranes, nuclear membranes and in mitochondria. In virus-infected cells, ATPase activity was also observed in cytoplasmic vesicles which were found in close proximity to the virus-specific cytoplasmic inclusion bodies (CI), at the ends of the arms of the CI and in plasmodesmata.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Potyvirus/fisiología , Zea mays/microbiología , Cuerpos de Inclusión Viral/enzimología , Enfermedades de las Plantas , Potyvirus/ultraestructura , ARN Viral/biosíntesis , Zea mays/enzimologíaRESUMEN
Attempts at overexpressing T4-phage deoxycytidylate deaminase using the pET3c/BL21(DE3)/pLysS system resulted in this enzyme being part of an inactive inclusion-body complex. However, by employing an enriched growth medium it was found that the deaminase could be induced in a soluble active form to at least 20% of this organism's cellular protein. Insoluble inclusion bodies were obtained with less rich media. This procedure was employed successfully with other highly expressed proteins that formed inclusion bodies. The use of a rich growth medium during the course of protein induction may be a valuable adjunct to limiting inclusion body formation with this as well as other expression systems.
Asunto(s)
Bacteriófago T4/enzimología , DCMP Desaminasa/biosíntesis , Cuerpos de Inclusión Viral/enzimología , Bacteriófago T4/genética , Secuencia de Bases , Clonación Molecular , Medios de Cultivo , DCMP Desaminasa/genética , DCMP Desaminasa/aislamiento & purificación , Escherichia coli/genética , Genes Virales/genética , Vectores Genéticos/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Estructurales Virales/genéticaRESUMEN
The properties of an endogenous RNA-dependent DNA polymerase (reverse transcriptase) associated with retrovirus-like particles (VLP) in a Drosophila melanogaster cell line were characterized. The enzyme requires a monovalent and a divalent cation, a sulfhydril-reducing agent and the four deoxynucleosides triphosphates for a maximal activity. The reaction was enhanced by a detergent and was sensitive to a DNA-A-free RNA-A indicating that the enzymatic activity was indeed associated with VLP which contain intrinsic RNA. The maximal incorporation of the labelled deoxynucleotide was observed at 25 degrees C in the presence of Mn2+. The enzyme responded well to exogenous template-primers in the similar way to that of retroviral reverse transcriptase and the use of several inhibitors confirms the presence of a real reverse transcriptase activity associated with virus-like particles in drosophila cells.
Asunto(s)
ADN Polimerasa Dirigida por ARN/metabolismo , Retroviridae/enzimología , Animales , Línea Celular , Drosophila melanogaster , Cuerpos de Inclusión Viral/enzimologíaRESUMEN
Sera from 367 patients presumed to have NANB hepatitis were screened for reverse transcriptase activity. In 29 cases significantly increased enzyme activities could be observed. In contrast, sera from 338 patients did not contain significant reverse transcriptase activities. 207 healthy individuals, 7 patients with hepatitis A and 6 patients with hepatitis B who served as controls were all negative for reverse transcriptase activity. The specificity of the enzyme assay was demonstrated by estimation of reverse transcriptase activity in sera from 10 "healthy" HIV-1-antibody positive individuals. In 3 out of 10 cases significant reverse transcriptase activity was observed associated with the human immunodeficiency virus. Our results indicate that the presence of particle-associated reverse transcriptase activity in serum from patients with NANB hepatitis is indicative of the presence of a retrovirus-like agent in these cases. However, the relatively low prevalence of reverse transcriptase positive cases associated with the NANB hepatitis makes it rather questionable whether this agent is a frequent and specific factor in the etiology of NANB hepatitis.
Asunto(s)
Hepatitis C/enzimología , Hepatitis Viral Humana/enzimología , ADN Polimerasa Dirigida por ARN/sangre , Infecciones por Retroviridae/enzimología , Síndrome de Inmunodeficiencia Adquirida/enzimología , VIH/enzimología , Humanos , Cuerpos de Inclusión Viral/enzimologíaRESUMEN
The time course of appearance of type I topoisomerase activity after the infection of mouse L cytoplasts by vaccinia virus was determined. When the enucleation procedure was carried out with unsynchronized cell cultures, a high level of host-cell-specific type I topoisomerase activity was found associated with the resulting cytoplasts. If cells were first synchronized by the two-cycle thymidine block method and then enucleated after release, the level of host type I topoisomerase activity was also high for S-phase-enucleated cells but was very low for cytoplasts prepared from cells previously synchronized and enucleated during either the G1 or the G2 phase. After the infection of G1-phase-enucleated cytoplasts with vaccinia virus, newly synthesized type I topoisomerase activity first appeared at about 3 h postinfection. Virosomes were isolated from the infected, synchronized cytoplasts and assayed for the presence of type I topoisomerase activity. The activity remained at the top of a sucrose gradient, well resolved from the virosome fraction, at the low salt levels (0.01 M KCl, 0.01 M Tris hydrochloride, pH 8.0) normally used in the course of virosome purification. If the sedimentation was at the higher salt concentration (0.15 M KCl) at which the enzyme shows optimal activity, type I topoisomerase cosedimented with the virosome fraction onto the sucrose gradient cushion. These results show that the type I topoisomerase activity dependent upon vaccinia virus infection may be detected with high sensitivity in G1-phase-enucleated cytoplasts. The association with virosomes is consistent with an involvement of topoisomerase activity either in DNA replication or in late transcription.
Asunto(s)
Replicación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Virus Vaccinia/crecimiento & desarrollo , Vaccinia/enzimología , Replicación Viral , Animales , Ciclo Celular , Citoplasma/enzimología , Cuerpos de Inclusión Viral/enzimología , Células L , RatonesRESUMEN
The RNA-dependent DNA-polymerase activity was studied in the postmicrosomal fraction and in the microsomal sediment of the liver of the newborn and adult Wistar rats. In the microsomal sediment of 4-6 day old rats the RNA-dependent DNA-polymerase activity was approximately by one order of magnitude higher than in that of 2 week old and adult rats. In the postmicrosomal fraction of 3 day old rats the RNA-dependent DNA-polymerase activity was also higher, but only by 30-35%, than in that of animals of the other age groups. Particles with density of 1.17 g/ml were found in the microsomal sediment of all studied animals. The characteristic morphology, sensitivity of the particle RNA-dependent DNA-polymerase activity to RNAse and the presence of reverse transcriptase allow for these particles to be referred to as retroviruses. A suggestion is put forward that the high intensity of reverse transcription during the early postnatal period can be due to still continuing processes of cell differentiation and enzymatic imprinting.
Asunto(s)
Cuerpos de Inclusión Viral/enzimología , Hígado/enzimología , ADN Polimerasa Dirigida por ARN/metabolismo , Retroviridae/enzimología , Animales , Animales Recién Nacidos , Hígado/crecimiento & desarrollo , Hígado/ultraestructura , Microsomas Hepáticos/enzimología , Ratas , Ratas EndogámicasRESUMEN
By a sensitive fluorometric assay method, we could definitely demonstrate neuraminidase activity for two variants of parainfluenza 3 virus, M and SC, which were previously shown to have no detectable neuraminidase activity. The enzyme activities of these viruses were very similar to each other, showing a much lower catalytic rate, a much higher Km value, and a more acidic pH optimum than those of the virus variants of high neuraminidase activity, 910N, LT, and MR. M and SC viruses eluted from guinea pig erythrocytes very poorly, whereas 910N and LT viruses eluted readily. M virus required the aid of a bacterial neuraminidase for effective growth and plaque formation in MDBK cells, but the virus grew well and formed plaques in R66 and Vero cells without the enzyme. SC virus required no exogenous neuraminidase for growth in all of these cell types. Depending on cell type, SC virus induced slight to extensive syncytial formation which was greatly inhibited by exogenous neuraminidase. In contrast, M virus induced extensive syncytial formation in all these cells regardless of the presence or absence of exogenous neuraminidase, although development and disintegration of the syncytia were more or less retarded by the enzyme, especially in MDBK cells. These results indicate that M virus possesses highly potent inducibility of syncytial formation which is further fortified by being low in viral neuraminidase activity.
Asunto(s)
Variación Genética , Cuerpos de Inclusión Viral/enzimología , Neuraminidasa/análisis , Virus de la Parainfluenza 3 Humana/enzimología , Respirovirus/enzimología , Adsorción , Animales , Fusión Celular , Efecto Citopatogénico Viral , Hemaglutinación por Virus , Humanos , Virus de la Parainfluenza 3 Humana/fisiología , Ensayo de Placa Viral , Cultivo de Virus , Replicación ViralRESUMEN
The role played by the gut juice of insects in the infective process of insect viruses was examined. Analysis of larval gut extract of Heliothis armigera by SDS-PAGE revealed protease activity associated with components of molecular weights 48,000 and 94,000. Proteases were found to be associated with occlusion bodies and virions of both nuclear polyhedrosis virus (NPV) and cytoplasmic polyhedrosis virus (CPV) infecting H. armigera. CPV occlusion bodies were dissolved by gut juice extract at pH 8.0, trypsin and chymotrypsin at pH 8.0, and carbonate-chloride solution at pH 10.5. Trypsin treatment was selective for occlusion bodies of CPV at pH 8.0, whereas solutions more alkaline than pH 10.0 without added enzymes were adequate to digest NPV occlusion bodies. This property was used to identify and separate the two types of viruses from a mixed infection. Gut extract proteases have characteristics similar to those of trypsin.