Development of a cell-based assay for monitoring specific hepatitis C virus NS3/4A protease activity in mammalian cells

Jin Ching Lee, Ya Feng Shih, Sung Po Hsu, Ten Yuan Chang, Lee Hua Chen, John T A Hsu

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

The hepatitis C virus (HCV) contains a positive-sense RNA genome that encodes a unique polyprotein precursor, which must be processed by proteases to enable viral maturation. Virally encoded NS3/4A protease has thus become an attractive target for the development of antiviral drugs. To establish an assay system for monitoring NS3/4A protease activity in mammalian cells, this study describes a substrate vector, pEG(Δ4AB)SEAP, in which enhanced green fluorescent protein (EGFP) was fused to secreted alkaline phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, Δ4AB, which spans the NS4A and NS4B junction region. Secretion of SEAP into the culture medium was demonstrated to depend on the cleavage of Δ4AB by HCV NS3/4A protease. We demonstrated that the accumulation of SEAP activity in the culture medium depends on time up to 60h with the coexpression of active NS3/4A protease. The amount of SEAP in the culture medium was around 10 times greater than that of cells with coexpression of inactive NS3/4A mutant protease. This strategy has made it possible to monitor NS3/4A activity inside mammalian cells. Moreover, by using cells containing the HCV subgenomic replicon, the EG(Δ4AB)SEAP reporter can be used to detect the anti-HCV activity of interferon-α (IFN-α). Consequently, this EG(Δ4AB)SEAP reporter can be used to screen for NS3/4A protease inhibitors in the cellular environment and for anti-HCV drugs in replicon cells.

Original languageEnglish
Pages (from-to)162-170
Number of pages9
JournalAnalytical Biochemistry
Volume316
Issue number2
DOIs
Publication statusPublished - May 15 2003
Externally publishedYes

Fingerprint

Viruses
Alkaline Phosphatase
Assays
Peptide Hydrolases
Cells
Monitoring
Hepacivirus
Culture Media
Replicon
Polyproteins
hepatitis C virus NS3 protein
Protease Inhibitors
Interferons
Antiviral Agents
Genes
Genome
RNA
Substrates
Pharmaceutical Preparations

Keywords

  • Antiviral
  • EGFP
  • Hepatitis C virus
  • NS3/4A protease
  • SEAP

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Development of a cell-based assay for monitoring specific hepatitis C virus NS3/4A protease activity in mammalian cells. / Lee, Jin Ching; Shih, Ya Feng; Hsu, Sung Po; Chang, Ten Yuan; Chen, Lee Hua; Hsu, John T A.

In: Analytical Biochemistry, Vol. 316, No. 2, 15.05.2003, p. 162-170.

Research output: Contribution to journalArticle

Lee, Jin Ching ; Shih, Ya Feng ; Hsu, Sung Po ; Chang, Ten Yuan ; Chen, Lee Hua ; Hsu, John T A. / Development of a cell-based assay for monitoring specific hepatitis C virus NS3/4A protease activity in mammalian cells. In: Analytical Biochemistry. 2003 ; Vol. 316, No. 2. pp. 162-170.
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