Biol. Chem., Vol. 392, pp. xxx-xxx, October 2011 • Copyright  by Walter de Gruyter • Berlin • Boston. DOI 10.1515/BC.2011.076 2011/143 Article in press - uncorrected proof 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, Stephanie A. Condotta, Jeremy Fenn, Andrea D. Olmstead and Francois Jean* ¸ Department of Microbiology and Immunology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada * Corresponding author e-mail: fjean@mail.ubc.ca Abstract The need to identify anti-Flaviviridae agents has resulted in intensive biochemical study of recombinant nonstructural (NS) viral proteases; however, experimentation on viral pro- tease-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 hepa- titis C virus (HCV) NS3-4A, plays a regulatory role in the substrate selectivity of the protease. In this study, we exam- ined trans-endoproteolytic cleavage activities of membrane- anchored and replicase-associated NS3-4A using an internally consistent set of membrane-anchored protein sub- strates 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 sub- strates. This stringent substrate recognition profile was also observed for the replicase-associated NS3-4A and is not gen- otype-specific. Our study also reveals that ER-anchoring of the substrate is critical for its cleavage by NS3-4A. Impor- tantly, 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 dis- covery and for the study of HCV biology. Keywords: cell-based assay; Flaviviridae protease; hepatitis C virus; induced-fit protease; membrane-anchored protease; viral protease. Introduction The hepatitis C virus (HCV) was identified in 1989 as the major causative agent of transfusion-associated non-A, non-B hepatitis (Choo et al., 1989). Much success has been achieved in understanding the mechanism by which this virus infects and replicates (Tang and Grise, 2009), infor- mation essential for the creation of vaccines and therapeutics (Pereira and Jacobson, 2009; Shimakami et al., 2009). HCV is a single-stranded positive-sense RNA virus belonging to the family Flaviviridae. Replication takes place in the cyto- plasm of liver cells (Sansonno et al., 1995); the endoplasmic reticulum (ER) is restructured by viral proteins, creating a mitochondria-associated ‘membranous web’ that houses the large macromolecular protein assemblies responsible for copy- ing the viral RNA (Moradpour et al., 2003). These ‘repli- case’ complexes contain non-structural (NS) viral enzymes (proteases, helicases, and polymerases), the activities as well as the maturation of which are promising drug targets. The HCV genome is a polycistronic RNA that is translated into one continuous polypeptide. The 10 proteins contained in this polyprotein (C-E1-E2-P7-NS2-NS3-NS4A-NS4B- NS5A-NS5B) are released by specific proteolytic cleavages mediated by both host and viral proteases (Dubuisson, 2007; Welbourn and Pause, 2007). One of the viral proteases, non- structural protein 3 (NS3), is involved in the release of itself as well as NS4A, NS4B, NS5A, and NS5B from the poly- protein. This protease has been shown to be essential for HCV replication in chimpanzees and has been the target of intensive study and inhibitor screening (Kolykhalov et al., 1994; Chen and Njoroge, 2009; Raney et al., 2010). NS3 is a bifunctional enzyme, containing both helicase and protease activities in distinct domains. The protease domain (NS3 pro ) is a chymotrypsin-like serine protease that acts in concert with its cofactor, NS4A (Morikawa et al., 2011). NS4A integrates into the NS3 protease fold (Kim et al., 1996, 1998), stabilizing and targeting NS3 to the ER by virtue of the N-terminal hydrophobic region in NS4A (Wolk et al., 2000). Through transient expression of HCV NS pre- cursors in mammalian cells, NS4A has been shown to increase NS3 proteolytic activity for the NS4A/NS4B and NS5A/NS5B junctions and to be essential for the cleavage of the NS4B/NS5A junction (Bartenschlager et al., 1994; Failla et al., 1994; Lin et al., 1994; Tanji et al., 1995). The region of NS4A required for this activation has been mapped to 13 central amino acids; a synthetic peptide corresponding to this region (pep4A) activates NS3 in vitro (Bartenschlager et al., 1995b; Steinkuhler et al., 1996b; Tomei et al., 1996; Richer et al., 2004). The NS3-NS4A heterocomplex (referred to hereafter as NS3-4A) cleavage site conforms to a consensus pattern of D/E-X-X-X-X-C/TxS/A (Grakoui et al., 1993). The trans cleavage sites (NS4A/NS4B, NS4B/NS5A, and NS5A/ AUTHOR’S COPY | AUTORENEXEMPLAR