In vivo determination of substrate specificity of hepatitis C virus NS3 protease: genetic assay for site-specific proteolysis.
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.
Kim
S Y
SY
Department of Life Science, Kwangju Institute of Science and Technology, Kwangju, 500-712, Korea.
Park
K W
KW
Lee
Y J
YJ
Back
S H
SH
Goo
J H
JH
Park
O K
OK
Jang
S K
SK
Park
W J
WJ
D000596:Amino Acids / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000378:metabolism; D015723:Gene Library; D017930:Genes, Reporter; D010455:Peptides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000596:Amino Acids; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010455:Peptides; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D017361:Viral Nonstructural Proteins; C052130:GAL4 protein, S cerevisiae; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases