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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred


IMPACT FACTOR 2018: 3.014
5-year IMPACT FACTOR: 3.162

CiteScore 2018: 3.09

SCImago Journal Rank (SJR) 2018: 1.482
Source Normalized Impact per Paper (SNIP) 2018: 0.820

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1437-4315
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Volume 392, Issue 10

Issues

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

Morgan M. Martin
  • Department of Microbiology and Immunology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada
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/ Stephanie A. Condotta
  • Department of Microbiology and Immunology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada
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/ Jeremy Fenn
  • Department of Microbiology and Immunology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada
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/ Andrea D. Olmstead
  • Department of Microbiology and Immunology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada
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/ François Jean
  • Department of Microbiology and Immunology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada
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Published Online: 2011-07-13 | DOI: https://doi.org/10.1515/BC.2011.076

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

About the article

Corresponding author


Received: 2011-04-30

Accepted: 2011-06-05

Published Online: 2011-07-13

Published in Print: 2011-10-01


Citation Information: Biological Chemistry, Volume 392, Issue 10, Pages 927–935, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/BC.2011.076.

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