Enzymatic characterization of membrane-associated hepatitis C virus NS3-4A heterocomplex serine protease activity expressed in human cells.
The hepatitis C virus (HCV) nonstructural (NS)3-NS4A serine protease heterocomplex is a prime target for development of novel HCV therapies, due to its essential role in maturation of the viral polyprotein. While the mode of substrate/inhibitor recognition of the HCV NS3/NS4A serine protease has been extensively studied in vitro, important molecular aspects of the mechanism of action for this membrane-bound multifunctional enzyme remain unresolved in vivo. In particular, what influence does membrane association exert on the specificity and catalysis of NS3-4A protease? To carry out this study, we developed a specific and sensitive protease assay using a unique internally quenched fluorogenic substrate (IQFS). Our IQFS enables for the first time the direct, specific detection of NS3-4A protease activity within membrane fractions isolated from human cells expressing NS3-4A and the determination of its steady-state kinetic parameters, which were found to be K(m) = 51 +/- 3 microM and k(cat) = 0.39 min(-1). We also show that our fluorescence-based bioassay can be used to evaluate specifically the potency and mode of action of NS3-4A directed inhibitors, such as in the case of a known NS3-4A substrate-analogue inhibitor (K(i) = 22 nM). Our results indicate that the membrane anchoring of NS3 by NS4A does not affect the substrate/inhibitor recognition by the NS3-4A protease domain. Further investigation may reveal whether membrane association could be important for regulating other enzymatic activities associated with NS3 (e.g., helicase and/or ATPase) and/or regulating the recently proposed cross-talk between the protease and helicase activities.
Hamill
Pamela
P
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.
Jean
François
F
D002384:Catalysis; D045744:Cell Line, Tumor; D002474:Cell-Free System / Q000201:enzymology / Q000821:virology; D002863:Chromogenic Compounds / Q000737:chemistry / Q000378:metabolism; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology / Q000821:virology; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013752:Tetracycline / Q000737:chemistry / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D002863:Chromogenic Compounds; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D009842:Oligopeptides; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D013752:Tetracycline; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus