The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that... more The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these fulllength HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.
Gold single crystals oriented to expose the (111) and (210) faces were investigated using electro... more Gold single crystals oriented to expose the (111) and (210) faces were investigated using electrochemical immittance spectroscopy (EIS) in aqueous solutions of HClO 4 and KF in the double layer region with the aim of identifying and explaining the frequency dispersion of interfacial capacitance known as constant phase angle (CPA) dispersion. Au(111) and Au(210) were chosen as representing the whole range of variance of electrochemical properties of Au(hkl) electrodes. Au(111) as the most uniform, microscopically smooth surface behaved with almost ideal capacitance in HClO 4 solutions in the whole potential range and also in KF solutions, in that case with the exception of potentials well positive to the potential of zero charge (pzc). Au(210) being microscopically the most corrugated surface displayed significant CPA dispersion in both electrolytes. In HClO 4 , dispersion on Au(210) occurred mostly in the potential region slightly positive to the potential of zero charge where the capacitance hump of the Helmholtz layer appeared. Analogous dispersion occurred on Au(210) in KF solutions only at high concentrations. The behaviour closest to ideal, dispersionless behaviour was always observed at sufficiently negative potentials. In KF solutions at positive potentials dispersion on both electrodes may be attributed to the adsorption (OH − , HF). In the intermediate potential range, close to the Helmholtz capacitance hump it can be attributed to solvent-metal interactions. Dispersion was lower in well conducting (concentrated) electrolytes and this suggests its geometrical nature is related possibly to the fractal pattern of the structured solvent.
The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that... more The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these fulllength HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.
Gold single crystals oriented to expose the (111) and (210) faces were investigated using electro... more Gold single crystals oriented to expose the (111) and (210) faces were investigated using electrochemical immittance spectroscopy (EIS) in aqueous solutions of HClO 4 and KF in the double layer region with the aim of identifying and explaining the frequency dispersion of interfacial capacitance known as constant phase angle (CPA) dispersion. Au(111) and Au(210) were chosen as representing the whole range of variance of electrochemical properties of Au(hkl) electrodes. Au(111) as the most uniform, microscopically smooth surface behaved with almost ideal capacitance in HClO 4 solutions in the whole potential range and also in KF solutions, in that case with the exception of potentials well positive to the potential of zero charge (pzc). Au(210) being microscopically the most corrugated surface displayed significant CPA dispersion in both electrolytes. In HClO 4 , dispersion on Au(210) occurred mostly in the potential region slightly positive to the potential of zero charge where the capacitance hump of the Helmholtz layer appeared. Analogous dispersion occurred on Au(210) in KF solutions only at high concentrations. The behaviour closest to ideal, dispersionless behaviour was always observed at sufficiently negative potentials. In KF solutions at positive potentials dispersion on both electrodes may be attributed to the adsorption (OH − , HF). In the intermediate potential range, close to the Helmholtz capacitance hump it can be attributed to solvent-metal interactions. Dispersion was lower in well conducting (concentrated) electrolytes and this suggests its geometrical nature is related possibly to the fractal pattern of the structured solvent.
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