Activity Of Japanese Encephalitis Virus NS3 Helicase And Inhibitors Screening

Japanese encephalitis virus (JEV), an important mosquito-borne zoonotic pathogen prevalented in South-East Asia, is a member of the Flavivirus genus in the family of Flaviviridae. In recent years, its endemic regions have a tendency to expand. To date, there is no efficient and specific antiviral therapeutic agent available to treat the disease.The NS3 is a bifunctional protein with both protease and helicase activity. The C-terminal two-thirds of JEV NS3, encoded by the 440 amino acids, contains catalytic domains for helicase, nucleoside 5’-triphosphatase (NTPase), as well as 5’-terminal RNA triphosphatase activities. The main function of NS3 helicase is to unwind the double strands genomic RNA forming in the process of virus replication. The unwinding reaction uses energy derived from NTP hydrolysis and found to be essential for the replication of virus, seemes to be a promising antiviral drug target.In this study, the helicase soluble protein was successfully expressed in E.coli and purified. Then methods for detecting ATP hydrolysis and nucleic acid unwinding activity were developed by luminescence and fluorescence resonance energy transfer (FRET) method. The concentration of enzyme, substrate, capture strand, ATP and divalet ions were optimized in the ATPase and helicase reaction respectively. On this basis, method of high throughput screening (HTS) of NS3 helicase inhibitors was established. Based on the helicase structure, a Specs library containging 230,000. compounds were screening by molecular docking and 41 compounds were selected for the inhibition analysis. The inhibitory activity of the 41 compounds to NS3 helicase were determined by the HTS established above, and two inhibitors were obtained. Then the antiviral effects of the two compounds were identified by western blot, indirect immunofluorescence assay, plaque assay and does-dependent assay. This study provides a high throughput screening method for NS3 helicase of JEV, and off erred two lead compounds for JEV drug development.1. Expression and purification of the JEV NS3 helicaseThe JEV helicase/NTPase domain was amplified by polymerase chain reaction (PCR) from the JEV cDNA clone. The product of PCR was digested by restriction endonuclease Nco I and Xho I, and then cloned into the expression vector pET-30a, pET-28a, etc. Then different expression conditions were explored, and the souble protein was obtained in pET-30a. The NS3 helicase in supernatant was purified by Ni2+ affinity column chromatography and gel filtration chromatography, and identified by SDS-PAGE and western blot assay.2. Method for ATPase activity assayThe Kinase-Glo Plus Luminescent Kinase Assay kit was employed to detect the amount ATP remaining in the reaction, and established the method for ATPase activity detection. Optimize the reaction conditions of ATPase, and found that the ATPase activity dependended on the divalent ions, such as Mg2+, Mn2+, Ca2+and Zn2+.3. Unwinding activity of helicaseBased on the FRET, method for unwinding activity detection was established. Two complementary DNA were synthesized as substrates, one was a 3’BHQ-2-labeled 22-mer DNA (5’-GGTTCTGAGGGTGGCGGTACTA-3’), and the other was a 5’Cy5-labeled 36-mer DNA (5’-TAGTACCGCCACCCTCAGAACCTTTTTTTTTTTTTT-3’). The impacts of helicase and substrate concentration, ATP concentration, capture strand concentration, divalent ions concentration, and reaction temperature on unwinding activity were evaluated by altering the corresponding conditions. And the optimal helicase reaction system has been established, which is 3μmol/L helicase protein, 200nmol/L fluorescent substrate,240nmol/L quenched substrate,2.4μmol/L capture strand,2mmol/L ATP, 10mmol/L Mn2+/15mmol/L Mg2+, and 37℃ incubated for 60min before detecting.4. High throughput screening of NS3 helicase inhibitorsBased on molecular docking method, the the binding activity between helicase structure and the 230,000 compounds from the Specs company were predicted, the potential 41 compounds were bought and screened on molecular level. Added the compounds in the helicase/NTPase reaction system, the inhibitory effect on the ATPase and unwinding activity were detected. Six compounds were found to have inhibitory effect to ATPase and four compounds inhibited unwinding activity, in which 2 compounds inhibited both ATPase and unwinding activity, and the half maximal (50%) inhibitory concentration (IC) of the compounds (IC50) were determined to 148umol/L and 150μmol/L, respectively.5. The antiviral effects on JEV replicationThe two compounds (Specs number:AK-968/40733793 and AD-970/43253453) possessing both ATPase and unwinding inhibitory activity were selected in the antiviral research. Used 5-25μmol/L of compounds to treat JEV, and then through western blot and indirect immunofluorescence assay to analysis the expression of the viral protein. The results showed that compared with the virus control, JEV protein in the treated group significantly decreased. Then the antiviral activity was further evaluated by plaque assay, and found that in the compounds treated group, the viral titer significantly decreased. Moreover in the 40μmol/L group, the viral titer was 1,000-fold declined. The results indicated that the two compounds have a significant inhibitory effect on the JEV, and within a certain range, the higher of the concentration, the better of the inhibitory effect. Through does-dependent assay, the effective compounds concentration which reduces viral-induced CPE by 50%(EC50) was determined to 25.67μmol/L and 23.50μmol/L, respectively. At the same time, the 50% cytotoxic concentration (CC50) of the two compounds was determined to 50μmol/L and 200μmol/L, respectively.