Methods for detecting ATP hydrolysis and nucleic acid unwinding of Japanese encephalitis virus NS3 helicase.
Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic pathogen that is prevalent in south-east Asia. Because there is no specific antiviral agent, JEV still causes a high rate of neurologic sequelae and mortality in humans. The helicase encoded by the NS3 gene of JEV has emerged recently as a novel antiviral target for treatment. In this study, a soluble recombinant JEV helicase protein was expressed and purified. Methods for detecting the ATP hydrolysis and nucleic acid unwinding activity were developed by luminescence and fluorescence resonance energy transfer (FRET). The concentrations of enzyme, substrate, capture strand, ATP, and divalent ions were optimised in the ATPase and helicase reactions. The feasibility of using these two methods for high-throughput screening of NS3 helicase inhibitors is discussed.
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Jin'e
J
Fang
Huan
H
Li
Guiqing
G
Peng
Shengbo
S
Cao
F Fu
FF
Zhen
Huanchun
H
Chen
Yunfeng
Y
Song
D000255:Adenosine Triphosphate / Q000378:metabolism*; D004265:DNA Helicases / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004664:Encephalitis Virus, Japanese / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D008163:Luminescent Measurements; D009696:Nucleic Acids / Q000378:metabolism*; D020365:RNA Helicases / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000255:Adenosine Triphosphate; D004265:DNA Helicases; D004791:Enzyme Inhibitors; D009696:Nucleic Acids; D020365:RNA Helicases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
ATPase activity; High-throughput screening (HTS); Japanese encephalitis virus (JEV); NS3 helicase; Unwinding activity