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In-cell selectivity profiling of membrane-anchored and replicase-associated hepatitis C virus NS3-4A protease reveals a common, stringent substrate recognition profile

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Published/Copyright: July 13, 2011
Biological Chemistry
From the journal Biological Chemistry Volume 392 Issue 10

Abstract

The need to identify anti-Flaviviridae agents has resulted in intensive biochemical study of recombinant nonstructural (NS) viral proteases; however, experimentation on viral protease-associated replication complexes in host cells is extremely challenging and therefore limited. It remains to be determined if membrane anchoring and/or association to replicase-membrane complexes of proteases, such as hepatitis C virus (HCV) NS3-4A, plays a regulatory role in the substrate selectivity of the protease. In this study, we examined trans-endoproteolytic cleavage activities of membrane-anchored and replicase-associated NS3-4A using an internally consistent set of membrane-anchored protein substrates mimicking all known HCV NS3-4A polyprotein cleavage sequences. Interestingly, we detected cleavage of substrates encoding for the NS4B/NS5A and NS5A/NS5B junctions, but not for the NS3/NS4A and NS4A/NS4B substrates. This stringent substrate recognition profile was also observed for the replicase-associated NS3-4A and is not genotype-specific. Our study also reveals that ER-anchoring of the substrate is critical for its cleavage by NS3-4A. Importantly, we demonstrate that in HCV-infected cells, the NS4B/NS5A substrate was cleaved efficiently. The unique ability of our membrane-anchored substrates to detect NS3-4A activity alone, in replication complexes, or within the course of infection, shows them to be powerful tools for drug discovery and for the study of HCV biology.

Keywords: cell-based assay; Flaviviridae protease; hepatitis C virus; induced-fit protease; membrane-anchored protease; viral protease
Corresponding author
Received: 2011-4-30
Accepted: 2011-6-5
Published Online: 2011-7-13
Published in Print: 2011-10-1

©2011 by Walter de Gruyter Berlin Boston

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  1. REVIEW
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  4. Conformational changes of the chaperone SecB upon binding to a model substrate – bovine pancreatic trypsin inhibitor (BPTI)
  5. Iron-catalyzed oxidation of Trp residues in low-density lipoprotein
  6. MOLECULAR MEDICINE
  7. Influenza A virus infection activates cholesterol sulfotransferase (SULT2B1b) in the lung of female C57BL/6 mice
  8. CELL BIOLOGY AND SIGNALING
  9. Lack of ADAM15 in mice is associated with increased osteoblast function and bone mass
  10. Improved lentiviral gene transfer into human embryonic stem cells grown in co-culture with murine feeder and stroma cells
  11. Ligand-independent activation of the arylhydrocarbon receptor by ETK (Bmx) tyrosine kinase helps MCF10AT1 breast cancer cells to survive in an apoptosis-inducing environment
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  13. Toxicity of Alzheimer's disease-associated Aβ peptide is ameliorated in a Drosophila model by tight control of zinc and copper availability
  14. PROTEOLYSIS
  15. In-cell selectivity profiling of membrane-anchored and replicase-associated hepatitis C virus NS3-4A protease reveals a common, stringent substrate recognition profile
  16. Analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus
https://doi.org/10.1515/BC.2011.076
Keywords for this article
cell-based assay; Flaviviridae protease; hepatitis C virus; induced-fit protease; membrane-anchored protease; viral protease

Articles in the same Issue

  1. REVIEW
  2. Disruption of virus-host cell interactions and cell signaling pathways as an anti-viral approach against influenza virus infections
  3. PROTEIN STRUCTURE AND FUNCTION
  4. Conformational changes of the chaperone SecB upon binding to a model substrate – bovine pancreatic trypsin inhibitor (BPTI)
  5. Iron-catalyzed oxidation of Trp residues in low-density lipoprotein
  6. MOLECULAR MEDICINE
  7. Influenza A virus infection activates cholesterol sulfotransferase (SULT2B1b) in the lung of female C57BL/6 mice
  8. CELL BIOLOGY AND SIGNALING
  9. Lack of ADAM15 in mice is associated with increased osteoblast function and bone mass
  10. Improved lentiviral gene transfer into human embryonic stem cells grown in co-culture with murine feeder and stroma cells
  11. Ligand-independent activation of the arylhydrocarbon receptor by ETK (Bmx) tyrosine kinase helps MCF10AT1 breast cancer cells to survive in an apoptosis-inducing environment
  12. Insulin modulates glucose-dependent insulinotropic polypeptide (GIP) secretion from enteroendocrine K cells in rats
  13. Toxicity of Alzheimer's disease-associated Aβ peptide is ameliorated in a Drosophila model by tight control of zinc and copper availability
  14. PROTEOLYSIS
  15. In-cell selectivity profiling of membrane-anchored and replicase-associated hepatitis C virus NS3-4A protease reveals a common, stringent substrate recognition profile
  16. Analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus
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