title: In-cell selectivity profiling of membrane-anchored and replicase-associated hepatitis C virus NS3-4A protease reveals a common, stringent substrate recognition profile.
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.
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.
authors.forename: Morgan M
authors.initials: MM
authors.lastname: Martin
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Condotta
authors.forename: Jeremy
authors.initials: J
authors.lastname: Fenn
authors.forename: Andrea D
authors.initials: AD
authors.lastname: Olmstead
authors.forename: François
authors.initials: F
authors.lastname: Jean
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D016174:Hepacivirus / Q000201:enzymology*; D006526:Hepatitis C / Q000821:virology*; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases