scholarly journals Efficient trans-Encapsidation of Hepatitis C Virus RNAs into Infectious Virus-Like Particles

2008 ◽  
Vol 82 (14) ◽  
pp. 7034-7046 ◽  
Author(s):  
Eike Steinmann ◽  
Christiane Brohm ◽  
Stephanie Kallis ◽  
Ralf Bartenschlager ◽  
Thomas Pietschmann
Keyword(s):  
Tissue Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Efficiency ◽  
Infectious Virus ◽  
Buoyant Density ◽  
Target Cells ◽  
Packaging Cell Line ◽  
Virus Like Particles ◽  
Rna Elements

ABSTRACT Recently, complete replication of hepatitis C virus (HCV) in tissue culture was established using the JFH1 isolate. To analyze determinants of HCV genome packaging and virion assembly, we developed a system that supports particle production based on trans-packaging of subgenomic viral RNAs. Using JFH1 helper viruses, we show that subgenomic JFH1 replicons lacking the entire core to NS2 coding region are efficiently encapsidated into infectious virus-like particles. Similarly, chimeric helper viruses with heterologous structural proteins trans-package subgenomic JFH1 replicons. Like authentic cell culture-produced HCV (HCVcc) particles, these trans-complemented HCV particles (HCVTCP) penetrate target cells in a CD81 receptor-dependent fashion. Since HCVTCP production was limited by competition between the helper and subgenomic RNA and to avoid contamination of HCVTCP stocks with helper viruses, we created HCV packaging cells. These cells encapsidate various HCV replicons with high efficiency, reaching infectivity titers up to 106 tissue culture infectious doses 50 per milliliter. The produced particles display a buoyant density comparable to HCVcc particles and can be propagated in the packaging cell line but support only a single-round infection in naïve cells. Together, this work demonstrates that subgenomic HCV replicons are assembly competent, thus excluding cis-acting RNA elements in the core-to-NS2 genomic region essential for RNA packaging. The experimental system described here should be helpful to decipher the mechanisms of HCV assembly and to identify RNA elements and viral proteins involved in particle formation. Similar to other vector systems of plus-strand RNA viruses, HCVTCP may prove valuable for gene delivery or vaccination approaches.

scholarly journals Novel Mutations in a Tissue Culture-Adapted Hepatitis C Virus Strain Improve Infectious-Virus Stability and Markedly Enhance Infection Kinetics

2011 ◽  
Vol 85 (8) ◽  
pp. 3978-3985 ◽  
Author(s):  
M. V. Pokrovskii ◽  
C. O. Bush ◽  
R. K. F. Beran ◽  
M. F. Robinson ◽  
G. Cheng ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Strain ◽  
Infectious Virus ◽  
Novel Mutations

scholarly journals Interaction of Hepatitis C Virus-Like Particles and Cells: a Model System for Studying Viral Binding and Entry

2002 ◽  
Vol 76 (18) ◽  
pp. 9335-9344 ◽  
Author(s):  
Miriam Triyatni ◽  
Bertrand Saunier ◽  
Padma Maruvada ◽  
Anthony R. Davis ◽  
Luca Ulianich ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Direct Method ◽  
Low Density Lipoprotein ◽  
Buoyant Density ◽  
Density Lipoprotein ◽  
Low Density ◽  
Dependent Manner ◽  
Virus Like Particles ◽  
Lps Binding

ABSTRACT Hepatitis C virus-like particles (HCV-LPs) containing the structural proteins of HCV H77 strain (1a genotype) was used as a model for HCV virion to study virus-cell interaction. HCV-LPs showed a buoyant density of 1.17 to 1.22 g/cm3 in a sucrose gradient and formed double-shelled particles 35 to 49 nm in diameter. Flow cytometry analysis by an indirect method (detection with anti-E2 antibody) and a direct method (use of dye-labeled HCV-LPs) showed that HCV-LPs binds to several human hepatic (primary hepatocytes, HepG2, HuH7, and NKNT-3) and T-cell (Molt-4) lines. HCV-LPs binding to cells occurred in a dose- and calcium-dependent manner and was not mediated by CD81. Scatchard plot analysis suggests the presence of two binding sites for HCV-LPs with high (Kd ∼1 μg/ml) and low (Kd ∼50 to 60 μg/ml) affinities of binding. Anti-E1 and -E2 antibodies inhibited HCV-LPs binding to cells. While preincubation of HCV-LPs with very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), or high-density lipoprotein (HDL) blocked its binding to cells, preincubation of cells with VLDL, LDL, HDL, or anti-LDL-R antibody did not. Confocal microscopy analysis showed that, after binding to cells, dye-labeled HCV-LPs were internalized into the cytoplasm. This process could be inhibited with anti-E1 or anti-E2 antibodies, suggesting that E1 and E2 proteins mediate HCV-LPs binding and, subsequently, their entry into cells. Altogether, our results indicate that HCV-LPs can be used to further characterize the mechanisms involved in the early steps of HCV infection.

Cellular Binding and Entry of Hepatitis C Virus Pseudotypes and Virus-like Particles Requires a Different Set of Cellular Co-receptors

2006 ◽  
Vol 44 (01) ◽  
Author(s):  
A Haberstroh ◽  
H Barth ◽  
EK Schnober ◽  
JM Pestka ◽  
HM Diepolder ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Like Particles

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 Cellular Determinants of Hepatitis C Virus Assembly, Maturation, Degradation, and Secretion

2007 ◽  
Vol 82 (5) ◽  
pp. 2120-2129 ◽  
Author(s):  
Pablo Gastaminza ◽  
Guofeng Cheng ◽  
Stefan Wieland ◽  
Jin Zhong ◽  
Wei Liao ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Assembly ◽  
Buoyant Density ◽  
Infectious Hepatitis ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Independent Manner ◽  
Viral Egress ◽  
Vldl Assembly

ABSTRACT Intracellular infectious hepatitis C virus (HCV) particles display a distinctly higher buoyant density than do secreted virus particles, suggesting that the characteristic low density of extracellular HCV particles is acquired during viral egress. We took advantage of this difference to examine the determinants of assembly, maturation, degradation, and egress of infectious HCV particles. The results demonstrate that HCV assembly and maturation occur in the endoplasmic reticulum (ER) and post-ER compartments, respectively, and that both depend on microsomal transfer protein and apolipoprotein B, in a manner that parallels the formation of very-low-density lipoproteins (VLDL). In addition, they illustrate that only low-density particles are efficiently secreted and that immature particles are actively degraded, in a proteasome-independent manner, in a post-ER compartment of the cell. These results suggest that by coopting the VLDL assembly, maturation, degradation, and secretory machinery of the cell, HCV acquires its hepatocyte tropism and, by mimicry, its tendency to persist.

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 Binding of Hepatitis C Virus-Like Particles Derived from Infectious Clone H77C to Defined Human Cell Lines

2002 ◽  
Vol 76 (3) ◽  
pp. 1181-1193 ◽  
Author(s):  
Sabine Wellnitz ◽  
Bettina Klumpp ◽  
Heidi Barth ◽  
Susumu Ito ◽  
Erik Depla ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Human Cell ◽  
Envelope Glycoprotein ◽  
Infectious Clone ◽  
Cell Interactions ◽  
Target Cells ◽  
Human Cell Lines ◽  
Glycoprotein E2

ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of viral entry and infection has been hampered by the inability to efficiently propagate the virus in cultured cells and the lack of a small-animal model. Recent studies have shown that in insect cells, the HCV structural proteins assemble into HCV-like particles (HCV-LPs) with morphological, biophysical, and antigenic properties similar to those of putative virions isolated from HCV-infected humans. In this study, we used HCV-LPs derived from infectious clone H77C as a tool to examine virus-cell interactions. The binding of partially purified particles to human cell lines was analyzed by fluorescence-activated cell sorting with defined monoclonal antibodies to envelope glycoprotein E2. HCV-LPs demonstrated dose-dependent and saturable binding to defined human lymphoma and hepatoma cell lines but not to mouse cell lines. Binding could be inhibited by monoclonal anti-E2 antibodies, indicating that the HCV-LP-cell interaction was mediated by envelope glycoprotein E2. Binding appeared to be CD81 independent and did not correlate with low-density lipoprotein receptor expression. Heat denaturation of HCV-LPs drastically reduced binding, indicating that the interaction of HCV-LPs with target cells was dependent on the proper conformation of the particles. In conclusion, our data demonstrate that insect cell-derived HCV-LPs bind specifically to defined human cell lines. Since the envelope proteins of HCV-LPs are presumably presented in a virion-like conformation, the binding of HCV-LPs to target cells may allow the study of virus-host cell interactions, including the isolation of HCV receptor candidates and antibody-mediated neutralization of binding.

scholarly journals Pol I-transcribed hepatitis C virus genome RNA replicates, produces an infectious virus and leads to severe hepatic steatosis in transgenic mice

2015 ◽  
Vol 36 (3) ◽  
pp. 159-167 ◽  
Author(s):  
Mohammod Johirul ISLAM ◽  
Keisuke HIKOSAKA ◽  
Hidenao NORITAKE ◽  
Mohammad Khaja Mafij UDDIN ◽  
Mohammed Badrul AMIN ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hepatic Steatosis ◽  
Infectious Virus ◽  
Virus Genome ◽  
Pol I ◽  
B Mice

scholarly journals Robust Production of Infectious Hepatitis C Virus (HCV) from Stably HCV cDNA-Transfected Human Hepatoma Cells

2005 ◽  
Vol 79 (22) ◽  
pp. 13963-13973 ◽  
Author(s):  
Zhaohui Cai ◽  
Chen Zhang ◽  
Kyung-Soo Chang ◽  
Jieyun Jiang ◽  
Byung-Chul Ahn ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Culture System ◽  
Infectious Virus ◽  
Hcv Infection ◽  
Hcv Rna ◽  
Human Hepatoma ◽  
Stable Cell Lines ◽  
Increased Risk

ABSTRACT Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide, with an increased risk of developing cirrhosis and hepatocellular carcinoma. The study of HCV replication and pathogenesis has been hampered by the lack of an efficient stable cell culture system and small-animal models of HCV infection and propagation. In an effort to develop a robust HCV infection system, we constructed stable human hepatoma cell lines that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus. Transcriptional expression of the full-length HCV RNA genome is under the control of a cellular Pol II polymerase promoter at the 5′ end and a hepatitis delta virus ribozyme at the 3′ end. The resulting HCV RNA was expressed and replicated efficiently, as shown by the presence of high levels of HCV proteins as well as both positive- and negative-strand RNAs in the stable Huh7 cell lines. Stable cell lines robustly produce HCV virions with up to 108 copies of HCV viral RNA per milliliter (ml) of the culture medium. Subsequent infection of naïve Huh7.5 cells with HCV released from the stable cell lines resulted in high levels of HCV proteins and RNAs. Additionally, HCV infection was inhibited by monoclonal antibodies specific to CD81 and the HCV envelope glycoproteins E1 and E2, and HCV replication was suppressed by alpha interferon. Collectively, these results demonstrate the establishment of a stable HCV culture system that robustly produces infectious virus, which will allow the study of each aspect of the entire HCV life cycle.

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