In vitro cleavage of hepatitis C virus polyprotein substrates by purified recombinant NS3 protease.
The non-structural protein NS3 of hepatitis C virus has been expressed in bacteria as a polyhistidine fusion protein which can be produced in a soluble form and easily purified by affinity chromatography. Using an in vitro transcription and translation system we have been able to demonstrate that this protein can proteolytically process substrate molecules derived from the non-structural region of the polyprotein. Using this assay system we have been able to optimize basic biochemical characteristics of the purified enzyme. Parallel experiments show that the full-length NS3 protein also possesses ATPase activity, indicating the bifunctional nature of the protein. In contrast, purified NS3 in which the predicted catalytic serine has been mutated loses protease but retains ATPase activity.
D'Souza
E D
ED
Biology Division, Wellcome Research Laboratories, Beckenham, Kent, UK.
Grace
K
K
Sangar
D V
DV
Rowlands
D J
DJ
Clarke
B E
BE
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D015533:Transcriptional Activation / Q000187:drug effects; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000251:Adenosine Triphosphatases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases