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Antimicrobial Agents and Chemotherapy, November 2009, p. 4825-4834, Vol. 53, No. 11
0066-4804/09/$08.00+0     doi:10.1128/AAC.00601-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Kao-Lu Pan,1 Jin-Ching Lee,2* Hsing-Wen Sung,1 Teng-Yuang Chang,3 and John T.-A. Hsu3,4*

Department of Chemical Engineering, National Tsing-Hua University, Hsin-Chu, Taiwan, Republic of China,1 Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China,2 Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China,3 Department of Biological Science and Technology, National Chiao Tung University, Hsin-chu, Taiwan, Republic of China4

Received 4 May 2009/ Returned for modification 19 June 2009/ Accepted 25 August 2009

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({Delta}4B5A)SEAP, that carries a dual reporter, EG({Delta}4B5A)SEAP. The EG({Delta}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 {Delta}4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/infection in the Huh7.5-EG({Delta}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({Delta}4B5A)SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG({Delta}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.

* Corresponding author. Mailing address: Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35, Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, Republic of China. Phone: 886-37-246-166, ext. 35717. Fax: 886-37-586-456. E-mail for J.-C. Lee: jclee{at}kmu.edu.tw. E-mail for J. T.-A. Hsu: tsuanhsu{at}nhri.org.tw

{triangledown} Published ahead of print on 31 August 2009.

Antimicrobial Agents and Chemotherapy, November 2009, p. 4825-4834, Vol. 53, No. 11
0066-4804/09/$08.00+0     doi:10.1128/AAC.00601-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Chockalingam, K., Simeon, R. L., Rice, C. M., Chen, Z. (2010). A cell protection screen reveals potent inhibitors of multiple stages of the hepatitis C virus life cycle. Proc. Natl. Acad. Sci. USA 107: 3764-3769 [Abstract] [Full Text]  


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