Production of infectious hepatitis C virus particles in three-dimensional cultures of the cell line carrying the genome-length dicistronic viral RNA of genotype 1b.

We show that a dicistronic hepatitis C virus (HCV) genome of genotype 1b supports the production and secretion of infectious HCV particles in two independent three-dimensional (3D) culture systems, the radial-flow bioreactor and the thermoreversible gelation polymer (TGP), but not in monolayer cultures. Immunoreactive enveloped particles, which are 50-60 nm in diameter and are surrounded by membrane-like structures, are observed in the culture medium as well as at the endoplasmic reticulum membranes and in dilated cytoplasmic cisternae in spheroids of Huh-7 cells. Infection of HCV particles is neutralized by anti-E2 antibody or patient sera that interfere with E2 binding to human cells. Finally, the utility of the 3D-TGP culture system for the evaluation of antiviral drugs is shown. We conclude that the replicon-based 3D culture system allows the production of infectious HCV particles. This system is a valuable tool in studies of HCV morphogenesis in a natural host cell environment.

[1]  M. Ait-goughoulte,et al.  Hepatitis C Virus-Like Particle Budding: Role of the Core Protein and Importance of Its Asp111 , 2003, Journal of Virology.

[2]  Toshiaki Maruyama,et al.  Complete Replication of Hepatitis C Virus in Cell Culture , 2005, Science.

[3]  S. Ray,et al.  Humoral immune response in acute hepatitis C virus infection. , 2005, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[4]  D. Lavillette,et al.  Monoclonal Antibody AP33 Defines a Broadly Neutralizing Epitope on the Hepatitis C Virus E2 Envelope Glycoprotein , 2005, Journal of Virology.

[5]  E. Blanchard,et al.  Hepatitis C Virus-Like Particle Morphogenesis , 2002, Journal of Virology.

[6]  R. Bartenschlager,et al.  Production of infectious hepatitis C virus in tissue culture from a cloned viral genome , 2005, Nature Medicine.

[7]  P. Barr,et al.  Genetic organization and diversity of the hepatitis C virus. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[8]  M. Manns,et al.  Early virologic response to treatment with peginterferon alfa‐2b plus ribavirin in patients with chronic hepatitis C , 2003, Hepatology.

[9]  C. Tinelli,et al.  Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: A case-control study , 1996 .

[10]  M. Nagaya,et al.  Evaluation of Thermoreversible Gelation Polymer for Regeneration of Focal Liver Injury , 2004, European Surgical Research.

[11]  V. Bichko,et al.  Effect of Alpha Interferon on the Hepatitis C Virus Replicon , 2001, Journal of Virology.

[12]  S. Mizutani,et al.  Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: Ultrafine structure of functionally enhanced hepatocarcinoma cell lines , 1998, In Vitro Cellular & Developmental Biology - Animal.

[13]  T. Wakita,et al.  Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient * , 2001, Journal of medical virology.

[14]  R. Bartenschlager,et al.  Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture , 2002, Journal of Virology.

[15]  Charles M. Rice,et al.  Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication , 2002, Journal of Virology.

[16]  P. Roingeard,et al.  Ultrastructural and physicochemical characterization of the hepatitis C virus recovered from the serum of an agammaglobulinemic patient , 1998, Archives of Virology.

[17]  S. Lemon,et al.  Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[18]  M. Mizokami,et al.  Genotype 2a Hepatitis C Virus Subgenomic Replicon Can Replicate in HepG2 and IMY-N9 Cells* , 2004, Journal of Biological Chemistry.

[19]  N. Kato,et al.  Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[20]  T. Matsuura,et al.  CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver , 2003, Hepatology.

[21]  T. Takato,et al.  Gene expression profile of human mesenchymal stem cells during osteogenesis in three-dimensional thermoreversible gelation polymer. , 2004, Biochemical and biophysical research communications.

[22]  R. Lamb,et al.  Escaping from the cell: assembly and budding of negative-strand RNA viruses. , 2004, Current topics in microbiology and immunology.

[23]  M. Mizokami,et al.  Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. , 2003, Gastroenterology.

[24]  S. Mizutani,et al.  High Density Culture of Immortalized Liver Endothelial Cells in the Radial-flow Bioreactor in the Development of an Artificial Liver , 1998, The International journal of artificial organs.

[25]  D. Wong,et al.  Hepatitis C Virus Structural Proteins Assemble into Viruslike Particles in Insect Cells , 1998, Journal of Virology.

[26]  M. Houghton,et al.  Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. , 1989, Science.

[27]  S. Lemon,et al.  Selectable Subgenomic and Genome-Length Dicistronic RNAs Derived from an Infectious Molecular Clone of the HCV-N Strain of Hepatitis C Virus Replicate Efficiently in Cultured Huh7 Cells , 2002, Journal of Virology.

[28]  Kenneth Koury,et al.  For Personal Use. Only Reproduce with Permission from the Lancet Publishing Group , 2022 .

[29]  J. Silver,et al.  Replication of Subgenomic Hepatitis C Virus Rnas in a Hepatoma Cell Line , 1999 .

[30]  M. Houghton,et al.  High titers of antibodies inhibiting the binding of envelope to human cells correlate with natural resolution of chronic hepatitis C , 1998, Hepatology.

[31]  A. Kasahara,et al.  Buoyant density of hepatitis C virus recovered from infected hosts: Two different features in sucrose equilibrium density‐gradient centrifugation related to degree of liver inflammation , 1994, Hepatology.

[32]  M. Houghton,et al.  A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[33]  N. Nakajima,et al.  Characterization of long-term cultures of hepatitis C virus , 1996, Journal of virology.

[34]  F. Miller,et al.  Significance of three-dimensional growth patterns of mammary tissues in collagen gels , 1983, In Vitro.

[35]  R. Bartenschlager,et al.  Characterization of Cell Lines Carrying Self-Replicating Hepatitis C Virus RNAs , 2001, Journal of Virology.

[36]  T. Takimoto,et al.  Molecular mechanism of paramyxovirus budding. , 2004, Virus research.

[37]  M. Houghton,et al.  Binding of hepatitis C virus to CD81. , 1998, Science.

[38]  N. Hayashi,et al.  In vitro binding of hepatitis C virus to CD81‐positive and ‐negative human cell lines , 2003, Journal of gastroenterology and hepatology.

[39]  P. André,et al.  Characterization of Low- and Very-Low-Density Hepatitis C Virus RNA-Containing Particles , 2002, Journal of Virology.

[40]  Tetsuro Suzuki,et al.  Quantitative Detection of Hepatitis C Virus (HCV) RNA in Saliva and Gingival Crevicular Fluid of HCV-Infected Patients , 2005, Journal of Clinical Microbiology.

[41]  M. Mondelli,et al.  Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: A prospective study , 1997, Hepatology.

[42]  M. Lai,et al.  Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts. , 2004, Virology.

[43]  G. Laurie,et al.  Basement membrane complexes with biological activity. , 1986, Biochemistry.

[44]  Masato Mikami,et al.  A Synthetic Hydrogel with Thermoreversible Gelation. I. Preparation and Rheological Properties , 1994 .

[45]  D. Burton,et al.  Robust hepatitis C virus infection in vitro. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[46]  C. Rice,et al.  Efficient initiation of HCV RNA replication in cell culture. , 2000, Science.

[47]  R. Purcell,et al.  Hepatitis C virus: Detection of intracellular virus particles by electron microscop , 1996 .

[48]  R. Compans.  Virus entry and release in polarized epithelial cells. , 1995, Current topics in microbiology and immunology.

[49]  R. Bartenschlager,et al.  Interferon-α inhibits hepatitis C virus subgenomic RNA replication by an MxA-independent pathway , 2001 .

[50]  D. Opstelten,et al.  Virus Maturation by Budding , 1998, Microbiology and Molecular Biology Reviews.

[51]  B. Bartosch,et al.  Cell entry of hepatitis C virus. , 2006, Virology.

[52]  Y. Matsuura,et al.  Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor. , 2003, Virology.

[53]  C. Rice,et al.  Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites , 1993, Journal of virology.