A reporter cell line for rapid and sensitive evaluation of hepatitis C virus infectivity and replication

Iro, Michaela; Witteveldt, Jeroen; Angus, Allan G. N.; Woerz, Ilka; Kaul, Artur; Bartenschlager, Ralf; Patel, Arvind H.

doi:10.1016/j.antiviral.2009.04.007

Cited by 48 publications

(65 citation statements)

References 36 publications

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“…To determine if these additional core mutations were responsible for the enhanced infectivity of JFH1 G33A seen during passaging, we individually engineered these second-site changes into the original JFH1 G33A genome. The viral replication levels of these mutants were tested using the Huh-7/J20 reporter cell line (22). This cell line stably expresses the enhanced green fluorescent protein fused in frame to the secreted alkaline phosphatase (SEAP) via a recognition sequence of the viral NS3/4A serine protease.…”

Section: Resultsmentioning

confidence: 99%

“…Human hepatoma Huh-7 and the Huh-7/ J20 reporter cell lines were propagated as described previously (22). The anti-NS5A mouse monoclonal antibody (MAb) 9E10 (26) was a kind gift from Charles M. Rice.…”

Section: Methodsmentioning

confidence: 99%

“…Virus titers in the supernatants were determined following immunostaining for NS5A by a 50% tissue culture infective dose (TCID 50 ) assay as described previously (26). The level of intracellular RNA replication was determined by measuring the secreted alkaline phosphatase (SEAP) activity in the culture medium as described previously (22). To monitor virus infectivity during serial passaging, 2 ϫ 10 6 Huh-7 cells were electroporated with 10 g of in vitro-transcribed viral RNA and seeded into a T80 flask in a total of 25 ml of medium.…”

Section: Methodsmentioning

confidence: 99%

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Conserved Glycine 33 Residue in Flexible Domain I of Hepatitis C Virus Core Protein Is Critical for Virus Infectivity

Angus

Loquet

Stack

et al. 2012

J Virol

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Hepatitis C virus core protein forms the viral nucleocapsid and plays a critical role in the formation of infectious particles. In this study, we demonstrate that the highly conserved residue G33, located within domain 1 of the core protein, is important for the production of cell culture-infectious virus (HCVcc). Alanine substitution at this position in the JFH1 genome did not alter viral RNA replication but reduced infectivity by ϳ2 logs. Virus production by this core mutant could be rescued by compensatory mutations located immediately upstream and downstream of the original G33A mutation. The examination of the helixloop-helix motif observed in the core protein structure (residues 15 to 41; Protein Data Bank entry 1CWX) indicated that the residues G33 and F24 are in close contact with each other, and that the G33A mutation induces a steric clash with F24. Molecular simulations revealed that the compensatory mutations increase the helix-loop-helix flexibility, allowing rescue of the core active conformation required for efficient virus production. Taken together, these data highlight the plasticity of core domain 1 conformation and illustrate the relationship between its structural tolerance to mutations and virus infectivity.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Conserved Glycine 33 Residue in Flexible Domain I of Hepatitis C Virus Core Protein Is Critical for Virus Infectivity

Angus

Loquet

Stack

et al. 2012

J Virol

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“…Human embryo kidney (HEK)-293T cells (ATCC CRL-1573) and the human hepatoma Huh-7 cells (43) were grown in Dulbecco modified Eagle medium (Invitrogen, United Kingdom) supplemented with 10% fetal calf serum, 100 U of penicillin/ml, 100 g of streptomycin/ml, 10 mM HEPES, 0.1 mM nonessential amino acids, and 2 mM glutamine. The secreted alkaline phosphatase (SEAP) reporter cell line Huh7J-20 was described previously (23).…”

Section: Methodsmentioning

confidence: 99%

Mutations within a Conserved Region of the Hepatitis C Virus E2 Glycoprotein That Influence Virus-Receptor Interactions and Sensitivity to Neutralizing Antibodies

Dhillon

Witteveldt

Gatherer

et al. 2010

J Virol

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Cell culture-adaptive mutations within the hepatitis C virus (HCV) E2 glycoprotein have been widely reported. We identify here a single mutation (N415D) in E2 that arose during long-term passaging of HCV strain JFH1-infected cells. This mutation was located within E2 residues 412 to 423, a highly conserved region that is recognized by several broadly neutralizing antibodies, including the mouse monoclonal antibody (MAb) AP33. Introduction of N415D into the wild-type (WT) JFH1 genome increased the affinity of E2 to the CD81 receptor and made the virus less sensitive to neutralization by an antiserum to another essential entry factor, SR-BI. Unlike JFH1 WT , the JFH1 N415D was not neutralized by AP33. In contrast, it was highly sensitive to neutralization by patient-derived antibodies, suggesting an increased availability of other neutralizing epitopes on the virus particle. We included in this analysis viruses carrying four other single mutations located within this conserved E2 region: T416A, N417S, and I422L were cell culture-adaptive mutations reported previously, while G418D was generated here by growing JFH1 WT under MAb AP33 selective pressure. MAb AP33 neutralized JFH1 T416A and JFH1 I422L more efficiently than the WT virus, while neutralization of JFH1 N417S and JFH1 G418D was abrogated. The properties of all of these viruses in terms of receptor reactivity and neutralization by human antibodies were similar to JFH1 N415D , highlighting the importance of the E2 412-423 region in virus entry.Hepatitis C virus (HCV), which belongs to the Flaviviridae family, has a positive-sense single-stranded RNA genome encoding a polyprotein that is cleaved by cellular and viral proteases to yield mature structural and nonstructural proteins. The structural proteins consist of core, E1 and E2, while the nonstructural proteins are p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (42). The hepatitis C virion comprises the RNA genome surrounded by the structural proteins core (nucleocapsid) and E1 and E2 (envelope glycoproteins). The HCV glycoproteins lie within a lipid envelope surrounding the nucleocapsid and play a major role in HCV entry into host cells (21). The development of retrovirus-based HCV pseudoparticles (HCVpp) (3) and the cell culture infectious clone JFH1 (HCVcc) (61) has provided powerful tools to study HCV entry.HCV entry is initiated by the binding of virus particles to attachment factors which are believed to be glycosaminoglycans (2), low-density lipoprotein receptor (41), and C-type lectins such as 37,38). Upon attachment at least four entry factors are important for particle internalization. These include CD81 (50), SR-BI (53) and the tight junction proteins claudin-1 (15) and occludin (6,36,51

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“…Mammalian cells stably expressing the EGFP-Delta4AB-SEAP cassette enabled the monitoring of NS3/4A activity upon expression in trans of the protease by subgenomic HCV replicon transfection. Iro et al [18], created a Huh7 cell line (designated Huh7-J20) expressing the identical cassette for rapid and sensitive quantification of HCV infection in cell culture by JFH-1 or JFH-1 chimeric viruses. Finally, Pan et al et al [19], generated a similar stable cell line (designated Huh7.5-EG(D4B5A)SEAP), which is based on the Huh-7.5 cells and the NS4B-NS5A junction region as a recognition sequence for the viral protease .…”

Section: Introductionmentioning

confidence: 99%

850 a Gaussia Luciferase Cell-Based System to Assess the Infection of Cell Culture- And Serum-Derived Hepatitis C Virus

Koutsoudakis¹,

Pulgar²,

González³

et al. 2012

Journal of Hepatology

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Robust replication of hepatitis C virus (HCV) in cell culture occurs only with the JFH-1 (genotype 2a) recombinant genome. The aim of this study was to develop a system for HCV infection quantification analysis and apply it for the selection of patient sera that may contain cell culture infectious viruses, particularly of the most clinically important genotype 1. Initially, a hepatoma cell line (designated Huh-7.5/EG(4A/4B)GLuc) was generated that stably expressed the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted Gaussia luciferase via a recognition sequence of the viral NS3/4A protease. Upon HCV infection, NS3/4A cleaved at its signal and the Gaussia was secreted to the culture medium, thus facilitating the infection quantification. The Huh-7.5/EG(4A/4B)GLuc cell line provided a rapid and highly sensitive quantification of HCV infection in cell culture using JFH-1-derived viruses. Furthermore, the Huh-7.5/EG(4A/4B)GLuc cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target distinct stages of the HCV life cycle; the Z-factor of this assay ranged from 0.72 to 0.75. Additionally, eighty-six sera derived from HCV genotype 1b infected liver transplant recipients were screened for their in vitro infection and replication potential. Approximately 12% of the sera contained in vitro replication-competent viruses, as deduced by the Gaussia signal, real time quantitative PCR, immunofluorescence and capsid protein secretion. We conclude that the Huh-7.5/EG(4A/4B)GLuc cell line is an excellent system not only for the screening of in vitro replication-competent serum-derived viruses, but also for the subsequent cloning of recombinant isolates. Additionally, it can be utilized for high-throughput screening of antiviral compounds.

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A reporter cell line for rapid and sensitive evaluation of hepatitis C virus infectivity and replication

Cited by 48 publications

References 36 publications

Conserved Glycine 33 Residue in Flexible Domain I of Hepatitis C Virus Core Protein Is Critical for Virus Infectivity

Conserved Glycine 33 Residue in Flexible Domain I of Hepatitis C Virus Core Protein Is Critical for Virus Infectivity

Mutations within a Conserved Region of the Hepatitis C Virus E2 Glycoprotein That Influence Virus-Receptor Interactions and Sensitivity to Neutralizing Antibodies

850 a Gaussia Luciferase Cell-Based System to Assess the Infection of Cell Culture- And Serum-Derived Hepatitis C Virus

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