In vivo determination of substrate specificity of hepatitis C virus NS3 protease: Genetic assay for site-specific proteolysis

Sung Yun Kim, Kye Won Park, Yong Jae Lee, Sung Hoon Back, Jae Hwan Goo, Ohkmae Kim, Sung Key Jang, Woo Jin Park

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Hepatitis C virus (HCV) NS3 protease is responsible for the processing of the viral polyprotein and is considered as a primary target for the development of anti-HCV therapy. We have developed a genetic method in yeast to screen for good substrate sequences of the NS3 protease. A library of fusion proteins was constructed with a transcription factor, GAL4, linked to the intracellular domain of an integral membrane protein, STE2, by a randomized protease substrate sequence. In yeast cells expressing NS3 protease, the substrate sequences in the fusion proteins were specifically recognized and cleaved. This cleavage resulted in the release of GAL4 from the cytoplasmic membrane and the subsequent activation of reporter genes by GAL4, which was detected by the growth of yeast cells on selective media. Based on the analysis of 69 isolated substrate sequences, a consensus sequence was deduced: (Glu/Asp)-X-Val-Val-(Leu/ Pro)-Cys ↓ (Ser/Ala), with the scissile bond being located between Cys and Ser or Ala and X not being determined. This is largely consistent with the previous results obtained by biochemical methods. An oligopeptide containing the deduced sequence was highly efficiently cleaved in vitro by the purified NS3 protease. These data demonstrated that the present genetic method could be used as an efficient tool for the in vivo determination of substrate specificity of proteases. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)42-48
Number of pages7
JournalAnalytical Biochemistry
Volume284
Issue number1
DOIs
Publication statusPublished - 2000 Aug 15
Externally publishedYes

Fingerprint

Proteolysis
Substrate Specificity
Assays
Peptide Hydrolases
Yeast
Substrates
Yeasts
valylvaline
Fusion reactions
Cells
Polyproteins
Oligopeptides
Consensus Sequence
Viperidae
Reporter Genes
Viruses
Hepacivirus
Libraries
Membrane Proteins
Proteins

Keywords

  • HCV
  • Protease
  • Randomized sequence
  • Saccharomyces cerevisae
  • Substrate specifity

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

In vivo determination of substrate specificity of hepatitis C virus NS3 protease : Genetic assay for site-specific proteolysis. / Kim, Sung Yun; Park, Kye Won; Lee, Yong Jae; Back, Sung Hoon; Goo, Jae Hwan; Kim, Ohkmae; Jang, Sung Key; Park, Woo Jin.

In: Analytical Biochemistry, Vol. 284, No. 1, 15.08.2000, p. 42-48.

Research output: Contribution to journalArticle

Kim, Sung Yun ; Park, Kye Won ; Lee, Yong Jae ; Back, Sung Hoon ; Goo, Jae Hwan ; Kim, Ohkmae ; Jang, Sung Key ; Park, Woo Jin. / In vivo determination of substrate specificity of hepatitis C virus NS3 protease : Genetic assay for site-specific proteolysis. In: Analytical Biochemistry. 2000 ; Vol. 284, No. 1. pp. 42-48.
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