Institute of Pathogen Biology, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, China.
PLoS One. 2012;7(1):e30259. doi: 10.1371/journal.pone.0030259. Epub 2012 Jan 20.
New therapeutic tools and molecular targets are needed for treatment of Japanese encephalitis virus (JEV) infections. JEV requires an α-1 translational frameshift to synthesize the NS1' protein required for viral neuroinvasiveness. Several flavonoids have been shown to possess antiviral activity in vitro against a wide spectrum of viruses. To date, the antiviral activities of flavonol kaempferol (Kae) and isoflavonoid daidzin (Dai) against JEV have not been described.
METHODOLOGY/PRINCIPAL FINDINGS: The 50% cytotoxic concentration (CC(50)) and 50% effective concentration (EC(50)) against JEV were investigated in BHK21 cells by MTS reduction. Activity against viral genomic RNA and proteins was measured by real-time RT-PCR and western blotting. The frameshift site RNA-binding characterization was also determined by electrospray ionization mass spectrometry, isothermal titration calorimetry and autodocking analysis. EC(50) values of Kae and Dai were 12.6 and 25.9 µM against JEV in cells pretreated before infection, whereas in cells infected before treatment, EC(50) was 21.5 and 40.4 µM, respectively. Kae exhibited more potent activity against JEV and RNA binding in cells following internalization through direct inhibition of viral replication and protein expression, indicating that its antiviral activity was principally due to direct virucidal effects. The JEV frameshift site RNA (fsRNA) was selected as a target for assaying Kae and Dai. ITC of fsRNA revealed an apparent K(b) value for Kae that was nine fold stronger than that for Dai. This binding was confirmed and localized to the RNA using ESI-MS and autodock analysis. Kae could form non-covalent complexes with fsRNA more easily than Dai could.
CONCLUSIONS/SIGNIFICANCE: Kae demonstrates more potent antiviral activity against JEV than does Dai. The mode of action of Kae as an anti-JEV agent seems to be related to its ability to inactivate virus by binding with JEV fsRNA.
需要新的治疗工具和分子靶点来治疗日本脑炎病毒（JEV）感染。JEV 需要一个α-1 翻译移码来合成病毒神经侵袭性所必需的 NS1'蛋白。已经证明，几种类黄酮在体外对广泛的病毒具有抗病毒活性。迄今为止，尚未描述类黄酮山柰酚（Kae）和异黄酮大豆苷元（Dai）对 JEV 的抗病毒活性。
方法/主要发现：通过 MTS 还原法在 BHK21 细胞中研究了对 JEV 的 50%细胞毒性浓度（CC（50））和 50%有效浓度（EC（50））。通过实时 RT-PCR 和 Western blot 测定对病毒基因组 RNA 和蛋白的活性。还通过电喷雾电离质谱、等温滴定量热法和自动对接分析确定了移码位点 RNA 结合特性。在感染前预处理细胞中，Kae 和 Dai 对 JEV 的 EC（50）值分别为 12.6 和 25.9 µM，而在感染前处理细胞中，EC（50）值分别为 21.5 和 40.4 µM。Kae 对 JEV 和 RNA 结合的活性更强，这表明其抗病毒活性主要归因于直接的杀病毒作用。JEV 移码位点 RNA（fsRNA）被选为测定 Kae 和 Dai 的靶标。fsRNA 的 ITC 显示出 Kae 的明显 K(b)值，是 Dai 的九倍。使用 ESI-MS 和自动对接分析证实并将其定位到 RNA。Kae 比 Dai 更容易与 fsRNA 形成非共价复合物。
结论/意义：Kae 对 JEV 的抗病毒活性比 Dai 更强。Kae 作为抗 JEV 药物的作用模式似乎与其通过与 JEV fsRNA 结合来失活病毒的能力有关。
