A reporter-based assay for identifying hepatitis C virus inhibitors based on subgenomic replicon cells

Lee, Jin‐Ching; Chang, Ching-Fung; Chi, Ya‐Hui; Hwang, Der-Ren; Hsu, John

doi:10.1016/j.jviromet.2003.10.007

Cited by 33 publications

(28 citation statements)

References 22 publications

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“…Human hepatoma cells (Huh-7) and HCV subgenomic-replicon cells (Ava5) were kindly provided by Apath, LLC (St. Louis, MO) (4). A reporter-based cell line, Ava5-EG(⌬4AB)SEAP (14,15), for HCV drug screening was derived from HCV replicon cells (Ava5). EG(⌬4AB)SEAP is a reporter gene expressing enhanced green fluorescent protein (EG), the NS3-NS4A protease decapeptide recognition sequence (⌬4AB), and secreted alkaline phosphatase (SEAP).…”

Section: Methodsmentioning

confidence: 99%

“…In the reporter cell line, a reporter gene, the EG(⌬4AB)SEAP gene, was stably integrated in the Ava5 cells to generate Ava5-EG(⌬4AB)SEAP cells. In Ava5-EG(⌬4AB)SEAP cells, SEAP activity in the culture medium can be used to reflect anti-HCV activity (14). Cells were maintained in a humidified atmosphere containing 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…Western blotting was performed essentially as described previously (14). Anti-HCV NS3 and antiactin antibodies were obtained from LTK Biolaboratories (Taipei, Taiwan) and CHEMICON International Inc. (Temecula, CA), respectively (10).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Inhibition of Hepatitis C Virus Replication by Antimonial Compounds

Hwang

Lin

Leu

et al. 2005

Antimicrob Agents Chemother

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Chronic hepatitis C virus (HCV) infection is a worldwide health problem causing serious complications, such as liver cirrhosis and hepatoma. Alpha interferon (IFN-␣) or its polyethylene glycol-modified form combined with ribavirin is the only recommended therapy. However, an alternative therapy is needed due to the unsatisfactory cure rate of the IFN-based therapy. Using a modified reporter assay based on the HCV subgenomic-replicon system, we found that sodium stibogluconate (SSG), a compound used for leishmania treatment, suppressed HCV replication. We have previously reported that SSG is effective at inhibiting HCV replication in a cell line permissive for HCV infection/replication and in an ex vivo assay using fresh human liver slices obtained from patients infected with HCV (26). In this study, we show that the SSG 50% inhibitory dose for HCV replication is 0.2 to 0.3 mg/ml (equivalent to 345 to 517 M of Sb) in the HCV subgenomicreplicon system. We also found that SSG and IFN-␣ exert a strong synergistic anti-HCV effect in both the traditional isobologram analysis and the median effect principle (CalcuSyn analysis). The combination of SSG and IFN-␣ could sustain the antiviral response better than SSG or IFN-␣ alone. The results suggest that SSG may be a good drug candidate for use in combination with other therapeutics, such as IFN-␣ and ribavirin, to treat HCV infection.Hepatitis C virus (HCV) causes a serious health problem worldwide (23). According to an estimate from the World Health Organization, approximately 3% of the world's population is infected by HCV (24). Although acute HCV infection is commonly associated with mild symptoms or is sometimes asymptomatic, most patients acquire a persistent infection that leads to severe chronic liver diseases, such as cirrhosis and hepatocellular carcinoma (13). Combination of ribavirin with alpha interferon (IFN-␣) (or its polyethylene glycol-modified form) is the only recommended therapy. However, this treatment achieves only an approximate 50% response rate to HCV genotype 1, the most prevalent genotype in HCV infections. Moreover, IFN-␣ and ribavirin combination therapy is expensive and often causes severe side effects (18). Given the high prevalence and severe clinical sequelae of HCV infection, there is an urgent need for the discovery and development of novel agents for HCV therapy.HCV represents the only genus (Hepacivirus) of the Flaviviridae virus family. HCV contains a single-stranded, positivesense RNA genome of 9.6 kb, which encodes a unique polyprotein of approximately 3,000 amino acids (5). The polyprotein is co-and posttranslationally processed by cellular and viral proteases to produce the mature structural and nonstructural viral proteins. The order of these proteins is NH 2 -C-E1-E2-P7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. Some HCV-encoded proteins can also be produced from alternate reading frames through ribosomal frameshifting (22, 25). One major obstacle impeding the development of HCV therapeutic strategies is the lack of a reproducible in vi...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Inhibition of Hepatitis C Virus Replication by Antimonial Compounds

Hwang

Lin

Leu

et al. 2005

Antimicrob Agents Chemother

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“…Cellular and viral proteases are involved in processing viral polyprotein into at least 10 proteins (core, E1, E2, p7, nonstructure protein 2 [NS2], NS3, NS4A, NS4B, NS5A, and NS5B), and the cleavages at the NS3-4A, NS4A-4B, NS4B-5A, and NS5A-5B junctions are mediated by NS3/4A protease (13,24,26). In the past few years, subgenomic HCV replicons have been employed extensively for studying viral replication, protein processing, and virus-host interactions and for discovering anti-HCV agents (4,15,16,29,34,52). However, such subgenomic systems are not useful for studying the entry, assembly, or release of viral particles, because they lack structure proteins.…”

mentioning

confidence: 99%

“…However, the associated common problem for these constructions is the low yield of viral progeny, possibly due to the insertion of undesirable elements that impair RNA replication. We have developed an efficient reporter-based assay for monitoring HCV protease activity and the degree of subgenomic replication (29)(30)(31). These studies involved the construction of a substrate vector, pEG(⌬4AB)SEAP, which contained a dual re-porter gene.…”

mentioning

confidence: 99%

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

Pan

Lee

Sung

et al. 2009

Antimicrob Agents Chemother

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A cell culture system for the production of hepatitis C virus (HCV) whole virions has greatly accelerated studies of the virus life cycle and the discovery of anti-HCV agents. However, the quantification of the HCV titers in a whole-virus infection/replication system currently relies mostly on reverse transcription-PCR or immunofluorescence assay, which would be cumbersome for high-throughput drug screening. To overcome this problem, this study has generated a novel cell line, Huh7.5-EG(⌬4B5A)SEAP, that carries a dual reporter, EG(⌬4B5A)SEAP. The EG(⌬4B5A)SEAP reporter is a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein is linked to secreted alkaline phosphatase (SEAP) in frame via ⌬4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/ infection in the Huh7.5-EG(⌬4B5A)SEAP cells can be quantitatively indicated by measuring the SEAP activity in cell culture medium. The levels of SEAP released from HCV-infected Huh7.5-EG(⌬4B5A)SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG(⌬4B5A)SEAP cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target multiple stages of the HCV life cycle. The Z-factor of this assay ranged from 0.64 to 0.74 in 96-well plates, indicating that this reporter system is suitable for high-throughput screening of prospective anti-HCV agents.

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InXy and SeXy, compact heterologous reporter proteins for mammalian cells

Fluri

Kelm

Lesage

et al. 2007

Biotech & Bioengineering

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Mammalian reporter proteins are essential for gene-function analysis, drugscreening initiatives and as model product proteins for biopharmaceutical manufacturing. Bacillus subtilis can maintain its metabolism by secreting Xylanase A (XynA), which converts xylan into shorter xylose oligosaccharides. XynA is a family 11 xylanase monospecific for D-xylose containing substrates. Mammalian cells transgenic for constitutive expression of wild-type xynA showed substantial secretion of this prokaryotic enzyme. Deletion analysis confirmed that a prokaryotic signal sequence encoded within the first 81 nucleotides was compatible with the secretory pathway of mammalian cells. Codon optimization combined with elimination of the prokaryotic signal sequence resulted in an exclusively intracellular mammalian Xylanase A variant (InXy) while replacement by an immunoglobulin-derived secretion signal created an optimal secreted Xylanase A derivative (SeXy). A variety of chromogenic and fluorescence-based assays adapted for use with mammalian cells detected InXy and SeXy with high sensitivity and showed that both reporter proteins resisted repeated freeze/thaw cycles, remained active over wide temperature and pH ranges, were extremely stable in human serum stored at room temperature and could independently be quantified in samples also containing other prominent reporter proteins such as the human placental alkaline phosphatase (SEAP) and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Glycoprofiling revealed that SeXy produced in mammalian cells was N- glycosylated at four different sites, mutation of which resulted in impaired secretion. SeXy was successfully expressed in a variety of mammalian cell lines and primary cells following transient transfection and transduction with adeno-associated virus particles (AAV) engineered for constitutive SeXy expression. Intramuscular injection of transgenic AAVs into mice showed significant SeXy levels in the bloodstream. InXy and SeXy are highly sensitive, compact and robust reporter proteins, fully compatible with pre-existing marker genes and can be assayed in high-throughput formats using very small sample volumes.

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A reporter-based assay for identifying hepatitis C virus inhibitors based on subgenomic replicon cells

Cited by 33 publications

References 22 publications

Inhibition of Hepatitis C Virus Replication by Antimonial Compounds

Inhibition of Hepatitis C Virus Replication by Antimonial Compounds

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

InXy and SeXy, compact heterologous reporter proteins for mammalian cells

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