Direct fluorometric measurement of hepatitis C virus helicase activity.
The non-structural protein 3 (NS3) of hepatitis C virus (HCV) is a highly promising target for anti-HCV therapy because of its multiple enzymatic activities, such as RNA-stimulated nucleoside triphosphatase, RNA helicase and serine protease. The helicase domain of NS3 as well as domain 2 of the helicase were expressed in a baculovirus system to obtain in high yield active proteins for prospective studies of complexes of the helicase with its inhibitors. A novel direct fluorometric test of helicase activity with a quenched DNA substrate, 3' labeled with a Cy3 dye and 5' labeled with a Black Hole Quencher, was developed and optimal reaction conditions established. This test based on fluorescence resonance energy transfer is simple and fast. It allows for direct measurements of enzyme activity, circumventing laborious and complicated radioactive techniques that are poorly reproducible. The results obtained encourage us to propose this new fluorescent assay as a method enabling high throughput screening of anti-helicase compounds.
Boguszewska-Chachulska
Anna M
AM
Institute of Biochemistry and Biophysics PAS, ul. Pawinskiego 5a, 02-106 Warsaw, Poland. annach@ibb.waw.pl
Krawczyk
Mariusz
M
Stankiewicz
Anna
A
Gozdek
Agnieszka
A
Haenni
Anne-Lise
AL
Strokovskaya
Ludmila
L
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D020134:Catalytic Domain; D002460:Cell Line; D005470:Fluorometry / Q000379:methods*; D016174:Hepacivirus / Q000201:enzymology*; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D008274:Magnesium; D008345:Manganese; D009841:Oligonucleotides; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases