Expression and purification of a hepatitis C virus NS3/4A complex, and characterization of its helicase activity with the Scintillation Proximity Assay system.
The C-terminal two-thirds of nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses RNA helicase activity. This enzyme is considered to be involved in viral replication, and is expected to be one of the target molecules of anti-HCV drugs. Previously, we established a high-throughput screening system for HCV helicase inhibitors using the Scintillation Proximity Assay (SPA) system [Kyono, K. et al. (1998) ANAL: BIOCHEM: 257, 120-126]. Here, we show improvement of the preparation method for the HCV NS3/4A complex. Alteration of the expression region led to an increase in protein expression. The partially purified full-length NS3 protein showed higher NS3 protease activity without the cofactor NS4A peptide than the truncated protease domain with the cofactor peptide, suggesting that this protein formed a complex with NS4A. NS3 further purified to homogeneity, as judged on silver staining, remained in a complex with NS4A. Characterization of the helicase activity of this full NS3/4A complex using the SPA helicase assay system revealed that this enzyme preferred Mn(2+), and that the optimal pH was 6.0-6.5. The NS3/4A complex could act on a DNA template but could not unwind the M13DNA/DNA substrate.
Kyono
Kiyoshi
K
Medicinal Chemistry Research Laboratories, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532-8505. k-kyono@tanabe.co.jp
Miyashiro
Masahiko
M
Taguchi
Ikuhiko
I
D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D009711:Nucleotides / Q000378:metabolism; D020365:RNA Helicases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012588:Scintillation Counting / Q000379:methods; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D002413:Cations, Divalent; D010450:Endopeptidases; D009711:Nucleotides; D020365:RNA Helicases; 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