Optimization of the multiple enzymatic activities of the hepatitis C virus NS3 protein.
The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is known to possess multiple enzymatic activities. In addition to its well-characterized protease activity, HCV NS3 also has ATP hydrolase (ATPase) and nucleic acid unwinding (helicase) activities. We systematically studied the effect of common reagents on all three enzymatic activities with a view to improving assay sensitivity for compound screening and profiling. Inclusion of the detergent lauryl dimethylamine oxide (LDAO) improves protease and helicase activities significantly, allowing robust assays at much lower NS3 concentrations. These conditions enable a particularly sensitive protease assay that uses picomolar concentrations of NS3.
Misialek
Shawn
S
InterMune, Brisbane, CA 94005, USA.
Rajagopalan
Ravi
R
Stevens
Sarah K
SK
Beigelman
Leonid
L
Seiwert
Scott D
SD
Kossen
Karl
K
D003902:Detergents / Q000494:pharmacology; D004123:Dimethylamines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016174:Hepacivirus / Q000201:enzymology*; D007700:Kinetics; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D003902:Detergents; D004123:Dimethylamines; D004791:Enzyme Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C014518:dodecyldimethylamine oxide