Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection

Pan, Kao-Lu; Lee, Jin‐Ching; Sung, Hsing‐Wen; Chang, Teng-Yuang; Hsu, John

doi:10.1128/aac.00601-09

Cited by 22 publications

(28 citation statements)

References 52 publications

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“…Several reports, including ours, have demonstrated that this model can overcome the limitations of the HCV replicon system and enable the discovery of compounds that target various stages of the HCV life cycle. [3][4][5][6][7][8] Using the HCV cell culture system, we previously developed a tube-capture-reverse transcription-polymerase chain reaction (RT-PCR) assay for screening HCV inhibitors and identified bisindolylmaleimides and indolocarbazoles as inhibitors of HCV replication. 4) Here, we describe another screening method for the detection of anti-HCV activity.…”

mentioning

confidence: 99%

A Cell-Based, Microplate Colorimetric Screen Identifies 7,8-Benzoflavone and Green Tea Gallate Catechins as Inhibitors of the Hepatitis C Virus

Fukazawa

Suzuki

Wakita

et al. 2012

Biological & Pharmaceutical Bulletin

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We describe a cell-based, microplate colorimetric screen for anti-hepatitis C virus (HCV) drugs that exploits the HCV-JFH1 viral culture system. Antiviral activity was assessed by measuring protection against the HCV-JFH1-induced cytopathic effect (CPE) in Huh7.5.1 cells using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability assay. The use of serum-free medium substantially sensitized Huh7.5.1 cells to HCV-induced CPE, causing sufficient cell death to perform colorimetric assays for anti-HCV activity in 96-well plates. As a proof of concept, we carried out a pilot screen of an inhibitor library and identified cyclosporin A and tamoxifen, two compounds with reported anti-HCV activity. Using the assay, we discovered the anti-HCV properties of the plant flavonoids epigallocatechin gallate (EGCG) and 7,8-benzoflavone (α-naphthoflavone). Other gallate-type catechins and flavones also displayed anti-HCV activity, but 5,6-benzoflavone (β-naphthoflavone), flavanone, and non-gallate catechins were inactive. EGCG apparently acted mainly on HCV entry, although it may also block other steps. In contrast, 7,8-benzoflavone was presumed to inhibit later stages of the HCV life cycle. This assay is simple, reliable and cost-effective; does not require any specially engineered cell lines or viruses; and should be useful in the identification of compounds with anti-HCV activity. Key words hepatitis C virus; 7,8-benzoflavone (α-naphthoflavone); epigallocatechin gallateMore than 170 million people worldwide are chronically infected with the hepatitis C virus (HCV) and are at risk for developing liver diseases such as cirrhosis and hepatocellular carcinoma. Vaccines against HCV are not currently available; furthermore, the standard interferon/ribavirin combination therapy is not effective in approximately half of HCV-infected patients, and it has considerable side effects.1,2) Thus, there is an obvious and urgent need for new agents that can enhance or replace current HCV therapies.Screening programs using HCV replicon-based systems have successfully identified compounds that act on HCV RNA replication. However, replicon systems do not reproduce the entire HCV life cycle, and they cannot isolate inhibitors of many important steps such as viral entry, assembly, and egress. HCV cell culture infection models that recapitulate the entire viral life cycle in vitro have greatly enhanced the opportunity for HCV drug discovery. Several reports, including ours, have demonstrated that this model can overcome the limitations of the HCV replicon system and enable the discovery of compounds that target various stages of the HCV life cycle. [3][4][5][6][7][8] Using the HCV cell culture system, we previously developed a tube-capture-reverse transcription-polymerase chain reaction (RT-PCR) assay for screening HCV inhibitors and identified bisindolylmaleimides and indolocarbazoles as inhibitors of HCV replication.4) Here, we describe another screening method for the detection of anti-HCV activity. This assay measures t...

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confidence: 99%

A Cell-Based, Microplate Colorimetric Screen Identifies 7,8-Benzoflavone and Green Tea Gallate Catechins as Inhibitors of the Hepatitis C Virus

Fukazawa

Suzuki

Wakita

et al. 2012

Biological & Pharmaceutical Bulletin

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“…Several recent reports describe systems that can in principle screen for inhibitors targeting different steps of the HCV life cycle via the prevention of the spread of cell culture-derived hepatitis C virus (HCVcc) infection in a cell monolayer (8)(9)(10)(11). Very recently, one such screen based on a colorimetric readout following immunostaining with an anti-E2 antibody identified inhibitors of multiple stages of the HCV life cycle (12).…”

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A cell protection screen reveals potent inhibitors of multiple stages of the hepatitis C virus life cycle

Chockalingam

Simeon

Rice

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The hepatitis C virus (HCV) life cycle involves multiple steps, but most current drug candidates target only viral replication. The inability to systematically discover inhibitors targeting multiple steps of the HCV life cycle has hampered antiviral development. We present a simple screen for HCV antivirals based on the alleviation of HCV-mediated cytopathic effect in an engineered cell linen4mBid. This approach obviates the need for a secondary screen to avoid cytotoxic false-positive hits. Application of our screen to 1280 compounds, many in clinical trials or approved for therapeutic use, yielded >200 hits. Of the 55 leading hits, 47 inhibited one or more aspects of the HCV life cycle by >40%. Six compounds blocked HCV entry to levels similar to an antibody (JS-81) targeting the HCV entry receptor CD81. Seven hits inhibited HCV replication and/or infectious virus production by >100-fold, with one (quinidine) inhibiting infectious virus production by 450-fold relative to HCV replication levels. This approach is simple and inexpensive and should enable the rapid discovery of new classes of HCV life cycle inhibitors.cell death | drug | high throughput | rescue | virus assembly H epatitis C virus (HCV) infection is a major public health problem, putting~180 million infected individuals worldwide (3% of the world's population) at risk for developing cirrhosis, hepatocellular carcinoma, and liver failure. Chronic hepatitis C is a leading cause for liver transplantation in the Western world. The current standard IFN-based therapy is costly, time-consuming, riddled with serious side effects, and cures only ∼50% of patients infected with the most common genotype. Although it is anticipated that small molecules that effectively inhibit the HCV life cycle can complement or even replace IFN therapy, no anti-HCV drugs have yet been approved for human use. Efforts to develop HCV antivirals mostly target only viral replication due to widespread dependence on the HCV replicon model (1-4), which effectively reproduces the RNA replication aspects of the HCV life cycle but does not allow screens to identify inhibitors of entry or infectious virus assembly and release.The identification of a genotype 2a isolate of HCV that reproduces the entire viral life cycle in cell culture (5-7) promised to accelerate the discovery of antivirals targeting all aspects of HCV growth in a more true-to-life model system. Several recent reports describe systems that can in principle screen for inhibitors targeting different steps of the HCV life cycle via the prevention of the spread of cell culture-derived hepatitis C virus (HCVcc) infection in a cell monolayer (8-11). Very recently, one such screen based on a colorimetric readout following immunostaining with an anti-E2 antibody identified inhibitors of multiple stages of the HCV life cycle (12). Although effective, this approach is labor-intensive and not readily amenable to high-throughput application, requiring antibodies and 12 washing steps in addition to a secondary screen to assess drug ...

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“…Pan et. al., while developing their similar dual reporter cell line Huh-7.5/EG(D4B5A)SEAP [19], checked various cleavable peptides as well, including the 4A/ 4B signal sequence utilized in this study. In an EGFP-SEAP configuration, the 4A/4B cleavage site appeared to be inaccessible by the viral protease.…”

Section: Discussionmentioning

confidence: 99%

“…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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Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection

Cited by 22 publications

References 52 publications

A Cell-Based, Microplate Colorimetric Screen Identifies 7,8-Benzoflavone and Green Tea Gallate Catechins as Inhibitors of the Hepatitis C Virus

A Cell-Based, Microplate Colorimetric Screen Identifies 7,8-Benzoflavone and Green Tea Gallate Catechins as Inhibitors of the Hepatitis C Virus

A cell protection screen reveals potent inhibitors of multiple stages of the hepatitis C virus life cycle

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

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