scholarly journals Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture

2010 ◽  
Vol 84 (21) ◽  
pp. 10999-11009 ◽  
Author(s):  
Pablo Gastaminza ◽  
Kelly A. Dryden ◽  
Bryan Boyd ◽  
Malcolm R. Wood ◽  
Mansun Law ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Structural Characteristics ◽  
Buoyant Density ◽  
Hcv Rna ◽  
Membrane Bilayer ◽  
Biophysical Characterization ◽  
Negative Stain ◽  
A Minor

ABSTRACT We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

scholarly journals Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly

2004 ◽  
Vol 78 (17) ◽  
pp. 9257-9269 ◽  
Author(s):  
Kevin C. Klein ◽  
Stephen J. Polyak ◽  
Jaisri R. Lingappa
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
De Novo ◽  
Signal Sequence ◽  
Buoyant Density ◽  
Capsid Assembly ◽  
Culture Systems ◽  
Capsid Formation ◽  
Cell Culture Systems

ABSTRACT The assembly of hepatitis C virus (HCV) is poorly understood, largely due to the lack of mammalian cell culture systems that are easily manipulated and produce high titers of virus. This problem is highlighted by the inability of the recently established HCV replicon systems to support HCV capsid assembly despite high levels of structural protein synthesis. Here we demonstrate that up to 80% of HCV core protein synthesized de novo in cell-free systems containing rabbit reticulocyte lysate or wheat germ extracts assembles into HCV capsids. This contrasts with standard primate cell culture systems, in which almost no core assembles into capsids. Cell-free HCV capsids, which have a sedimentation value of ≈100S, have a buoyant density (1.28 g/ml) on cesium chloride similar to that of HCV capsids from other systems. Capsids produced in cell-free systems are also indistinguishable from capsids isolated from HCV-infected patient serum when analyzed by transmission electron microscopy. Using these cell-free systems, we show that HCV capsid assembly is independent of signal sequence cleavage, is dependent on the N terminus but not the C terminus of HCV core, proceeds at very low nascent chain concentrations, is independent of intact membrane surfaces, and is partially inhibited by cultured liver cell lysates. By allowing reproducible and quantitative assessment of viral and cellular requirements for capsid formation, these cell-free systems make a mechanistic dissection of HCV capsid assembly possible.

scholarly journals Development of hepatitis C virus production reporter-assay systems using two different hepatoma cell lines

2012 ◽  
Vol 93 (7) ◽  
pp. 1422-1431 ◽  
Author(s):  
Midori Takeda ◽  
Masanori Ikeda ◽  
Yasuo Ariumi ◽  
Takaji Wakita ◽  
Nobuyuki Kato
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Hepatoma Cell ◽  
Rna Replication ◽  
Hcv Rna ◽  
Reporter Assay ◽  
Hepatoma Cell Lines

A hepatitis C virus (HCV) infection system was developed previously using the HCV JFH-1 strain (genotype 2a) and HuH-7 cells, and this cell culture is so far the only robust production system for HCV. In patients with chronic hepatitis C, the virological effects of pegylated interferon and ribavirin therapy differ depending on the HCV strain and the genetic background of the host. Recently, we reported the hepatoma-derived Li23 cell line, in which the JFH-1 life cycle is reproduced at a level almost equal to that in HuH-7-derived RSc cells. To monitor the HCV life cycle more easily, we here developed JFH-1 reporter-assay systems using both HuH-7- and Li23-derived cell lines. To identify any genetic mutations by long-term cell culture, HCV RNAs in HuH-7 cells were amplified 130 days after infection and subjected to sequence analysis to find adaptive mutation(s) for robust virus replication. We identified two mutations, H2505Q and V2995L, in the NS5B region. V2995L but not H2505Q enhanced JFH-1 RNA replication. However, we found that H2505Q but not V2995L enhanced HCV RNA replication of strain O (genotype 1b). We also selected highly permissive D7 cells by serial subcloning of Li23 cells. The expression levels of claudin-1 and Niemann–Pick C1-like 1 in D7 cells are higher than those in parental Li23 cells. In this study, we developed HCV JFH-1 reporter-assay systems using two distinct hepatoma cell lines, HuH-7 and Li23. The mutations in NS5B resulted in different effects on strains O and JFH-1 HCV RNA replication.

scholarly journals Comprehensive Analysis of the Effects of Ordinary Nutrients on Hepatitis C Virus RNA Replication in Cell Culture

2007 ◽  
Vol 51 (6) ◽  
pp. 2016-2027 ◽  
Author(s):  
Masahiko Yano ◽  
Masanori Ikeda ◽  
Ken-ichi Abe ◽  
Hiromichi Dansako ◽  
Shogo Ohkoshi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Vitamin D ◽  
Hepatitis C Virus ◽  
Linoleic Acid ◽  
Hepatitis C ◽  
Combined Treatment ◽  
Rna Replication ◽  
Assay System ◽  
Hcv Rna ◽  
Β Carotene

ABSTRACT To date, only a limited number of studies have reported finding an influence of ordinary nutrients on hepatitis C virus (HCV) RNA replication. However, the effects of other nutrients on HCV RNA replication remain largely unknown. We recently developed a reporter assay system for genome-length HCV RNA replication in hepatoma-derived HuH-7 cells (OR6). Here, using this OR6 assay system, we comprehensively examined 46 nutrients from four nutrient groups: vitamins, amino acids, fatty acids, and salts. We found that three nutrients—β-carotene, vitamin D2, and linoleic acid—inhibited HCV RNA replication and that their combination caused additive and/or synergistic effects on HCV RNA replication. In addition, combined treatment with each of the three nutrients and interferon alpha or beta or fluvastatin inhibited HCV RNA replication in an additive manner, while combined treatment with cyclosporine synergistically inhibited HCV RNA replication. In contrast, we found that vitamin E enhanced HCV RNA replication and negated the effects of the three anti-HCV nutrients and cyclosporine but not those of interferon or fluvastatin. These results will provide useful information for the treatment of chronic hepatitis C patients who also take anti-HCV nutrients as an adjunctive therapy in combination with interferon. In conclusion, among the ordinary nutrients tested, β-carotene, vitamin D2, and linoleic acid possessed anti-HCV activity in a cell culture system, and these nutrients are therefore considered to be potential candidates for enhancing the effects of interferon therapy.

scholarly journals Reduction of Hepatitis C Virus NS5A Hyperphosphorylation by Selective Inhibition of Cellular Kinases Activates Viral RNA Replication in Cell Culture

2004 ◽  
Vol 78 (23) ◽  
pp. 13306-13314 ◽  
Author(s):  
Petra Neddermann ◽  
Manuela Quintavalle ◽  
Chiara Di Pietro ◽  
Angelica Clementi ◽  
Mauro Cerretani ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Kinase Inhibitors ◽  
Rna Replication ◽  
Hcv Rna ◽  
Adaptive Mutations ◽  
Direct Demonstration ◽  
Huh7 Cells ◽  
Efficient Replication

ABSTRACT Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations. In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations. The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase(s) below a certain threshold enables HCV replication. Upon screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro and the formation of NS5A p58 in cell culture. Cells, transfected with the HCV Con1 wild-type sequence, support HCV RNA replication upon addition of any of the three compounds. The effect of the kinase inhibitors was found to be synergistic with coadaptive mutations in NS3. This is the first direct demonstration that the presence of high amounts of NS5A-p58 causes inhibition of HCV RNA replication in cell culture and that this inhibition can be relieved by kinase inhibitors.

scholarly journals Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture

2003 ◽  
Vol 77 (5) ◽  
pp. 3181-3190 ◽  
Author(s):  
Keril J. Blight ◽  
Jane A. McKeating ◽  
Joseph Marcotrigiano ◽  
Charles M. Rice
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Replication ◽  
Full Length ◽  
Hcv Rna ◽  
Metabolic Labeling ◽  
Virus Genotype ◽  
Adaptive Mutations ◽  
Genotype 1A

ABSTRACT Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-resistant liver disease worldwide. Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from genotype 1b isolates. Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77. Replication of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3 and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and full-length RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3 structure. The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for antiviral development. These results are also encouraging for the development of adapted replicons for the remaining HCV genotypes.

scholarly journals Production of Infectious Hepatitis C Virus of Various Genotypes in Cell Cultures

2007 ◽  
Vol 81 (9) ◽  
pp. 4405-4411 ◽  
Author(s):  
Takanobu Kato ◽  
Takuya Matsumura ◽  
Theo Heller ◽  
Satoru Saito ◽  
Ronda K. Sapp ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Culture Medium ◽  
Expression System ◽  
Core Antigen ◽  
Hcv Rna ◽  
Transfected Cells

ABSTRACT A unique hepatitis C virus (HCV) strain JFH-1 has been shown to replicate efficiently in cell culture with production of infectious HCV. We previously developed a DNA expression system containing HCV cDNA flanked by two self-cleaving ribozymes to generate HCV particles in cell culture. In this study, we produced HCV particles of various genotypes, including 1a (H77), 1b (CG1b), and 2a (J6 and JFH-1), in the HCV-ribozyme system. The constructs also contain the secreted alkaline phosphatase gene to control for transfection efficiency and the effects of culture conditions. After transfection into the Huh7-derived cell line Huh7.5.1, continuous HCV replication and secretion were confirmed by the detection of HCV RNA and core antigen in the culture medium. HCV replication levels of strains H77, CG1b, and J6 were comparable, whereas the JFH-1 strain replicates at a substantially higher level than the other strains. To evaluate the infectivity in vitro, the culture medium of JFH-1-transfected cells was inoculated into naive Huh7.5.1 cells. HCV proteins were detected by immunofluorescence 3 days after inoculation. To evaluate the infectivity in vivo, the culture medium from HCV genotype 1b-transfected cells was inoculated into a chimpanzee and caused a typical course of HCV infection. The HCV 1b propagated in vitro and in vivo had sequences identical to those of the HCV genomic cDNA used for cell culture transfection. The development of culture systems for production of various HCV genotypes provides a valuable tool not only to study the replication and pathogenesis of HCV but also to screen for antivirals.

scholarly journals Efficient infection of tree shrew (Tupaia belangeri) with hepatitis C virus grown in cell culture or from patient plasma

2007 ◽  
Vol 88 (9) ◽  
pp. 2504-2512 ◽  
Author(s):  
Xinping Xu ◽  
Hongbo Chen ◽  
Xiaomei Cao ◽  
Kunlong Ben
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Tree Shrew ◽  
Small Animal ◽  
Cost Effective ◽  
Hcv Rna ◽  
Tree Shrews ◽  
Huh7 Cells ◽  
Before And After

The generation of a new, cost-effective, non-primate, small-animal model would greatly facilitate research into hepatitis C virus (HCV) pathogenesis and the development of novel therapeutic and preventative technologies to control the increasing HCV threat to public health. Native HCV from patient plasma and HCV grown in cell culture (HCVcc) were used to inoculate adult tree shrews. Each animal was inoculated with one HCV genotype. Alanine aminotransferase (ALT) levels, HCV RNA and viral load were determined in the animals before and after inoculation. For native HCV, 16/18 inoculated tree shrews (89 %) became infected; 12/16 (75 %) of these animals became chronically infected, whilst infection was resolved in the remaining four (25 %). For HCVcc, infection occurred in 10/12 inoculated tree shrews (83 %) and chronic infection was observed in two of these animals. HCVcc from Huh7 cells showed a higher infectivity than that from HeLa cells. The animals inoculated with inadequate amounts of HCV were not infected in either native HCV or HCVcc experiments. Peak viral loads reached 103–105 international units ml−1 in chronically infected animals. ALT level changes reflected the normal fluctuation range in most animals. Thus, tree shrews without immunosuppression can be infected efficiently by native HCV and HCVcc when the animal is inoculated with an adequate amount of single-genotype HCV.

scholarly journals Endocannabinoid CB1 antagonists inhibit hepatitis C virus production, providing a novel class of antiviral host-targeting agents

2014 ◽  
Vol 95 (11) ◽  
pp. 2468-2479 ◽  
Author(s):  
Mahsa Shahidi ◽  
Enoch S. E. Tay ◽  
Scott A. Read ◽  
Mehdi Ramezani-Moghadam ◽  
Kazuaki Chayama ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Lipid Metabolism ◽  
Hepatitis C ◽  
Cannabinoid Receptor ◽  
Virus Production ◽  
Hcv Rna ◽  
Virus Infectivity ◽  
Subgenomic Replicon ◽  
Cb1 Antagonists

Direct-acting antivirals have significantly improved treatment outcomes in chronic hepatitis C (CHC), but side effects, drug resistance and cost mean that better treatments are still needed. Lipid metabolism is closely linked with hepatitis C virus (HCV) replication, and endocannabinoids are major regulators of lipid homeostasis. The cannabinoid 1 (CB1) receptor mediates these effects in the liver. We have previously shown upregulation of CB1 receptors in the livers of patients with CHC, and in a HCV cell-culture model. Here, we investigated whether CB1 blockade inhibited HCV replication. The antiviral effect of a CB1 antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), was examined in HCV strain JFH1 cell-culture and subgenomic replicon models. The effects on the expression of genes involved in lipid metabolism were also measured. CB1 short hairpin RNA (shRNA) was used to confirm that the effects were specific for the cannabinoid receptor. Treatment with AM251 strongly inhibited HCV RNA (~70 %), viral protein (~80 %), the production of new virus particles (~70 %) and virus infectivity (~90 %). As expected, AM251 reduced the expression of pro-lipogenic genes (SREBP-1c, FASN, SCD1 and ACC1) and stimulated genes promoting lipid oxidation (CPT1 and PPARα). This effect was mediated by AMP-activated protein kinase (AMPK). Stable CB1 knockdown of cells infected with HCV showed reduced levels of HCV RNA compared with controls. Thus, reduced CB1 signalling inhibits HCV replication using either pharmacological inhibitors or CB1 shRNA. This may be due, at least in part, to reduced lipogenesis, mediated by AMPK activation. We suggest that CB1 antagonists may represent an entirely new class of drug with activity against HCV.

scholarly journals Dominant negative effect of wild-type NS5A on NS5A-adapted subgenomic hepatitis C virus RNA replicon

2004 ◽  
Vol 85 (7) ◽  
pp. 1867-1875 ◽  
Author(s):  
Rita Graziani ◽  
Giacomo Paonessa
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Hcv Rna ◽  
Wild Type ◽  
Subgenomic Replicon ◽  
Adaptive Mutations ◽  
Negative Effect

An efficient model is currently used to study hepatitis C virus (HCV) replication in cell culture. It involves transfection in Huh7, a hepatoma-derived cell line, of an antibiotic (neomycin) selectable HCV subgenomic replicon encoding the non-structural (NS) proteins from NS3 to NS5B. However, strong and sustained replication is achieved only on the appearance of adaptive mutations in viral proteins. The most effective of these adaptive mutations are concentrated mainly in NS5A, not only into the original Con1 but also in the recently established HCV-BK and HCV-H77 isolate-derived replicons. This suggests that the expression of wild-type (wt) NS5A may not allow efficient HCV RNA replication in cell culture. With the use of a β-lactamase reporter gene as a marker for HCV replication and TaqMan RNA analysis, the replication of different HCV replicons in cotransfection experiments was investigated. Comparing wt with NS5A-adapted replicons, the strong evidence accumulated showed that the expression of wt NS5A was actually able to inhibit the replication of NS5A-adapted replicons. This feature was characterized as a dominant negative effect. Interestingly, an NS5B (R2884G)-adapted replicon, containing a wt NS5A, was dominant negative on an NS5A-adapted replicon but was not inhibited by the original Con1 replicon. In conclusion, these studies revealed that the original wt Con1 replicon is not only incompetent for replication in cell culture, but is also able to interfere with NS5A-adapted replicons.

scholarly journals Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture

2002 ◽  
Vol 76 (8) ◽  
pp. 4008-4021 ◽  
Author(s):  
Thomas Pietschmann ◽  
Volker Lohmann ◽  
Artur Kaul ◽  
Nicole Krieger ◽  
Gabriele Rinck ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hepatoma Cell ◽  
Buoyant Density ◽  
Full Length ◽  
Structural Proteins ◽  
Hepatoma Cell Line ◽  
Particle Assembly ◽  
Adaptive Mutations

ABSTRACT 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 full-length 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.

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