Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Madrid, 28668, Spain.
Life Science & Bioengineering Building, Technical University of Denmark, 2800, Kongens Lyngby, Denmark.
Sci Rep. 2019 Apr 1;9(1):5397. doi: 10.1038/s41598-019-41998-1.
Zika virus (ZIKV) is an emerging pathogen that has been associated with large numbers of cases of severe neurologic disease, including Guillain-Barré syndrome and microcephaly. Despite its recent establishment as a serious global public health concern there are no licensed therapeutics to control this virus. Accordingly, there is an urgent need to develop methods for the high-throughput screening of antiviral agents. We describe here a fluorescence-based method to monitor the real-time polymerization activity of Zika virus RNA-dependent RNA polymerase (RdRp). By using homopolymeric RNA template molecules, de novo RNA synthesis can be detected with a fluorescent dye, which permits the specific quantification and kinetics of double-strand RNA formation. ZIKV RdRp activity detected using this fluorescence-based assay positively correlated with traditional assays measuring the incorporation of radiolabeled nucleotides. We also validated this method as a suitable assay for the identification of ZIKV inhibitors targeting the viral polymerase using known broad-spectrum inhibitors. The assay was also successfully adapted to detect RNA polymerization activity by different RdRps, illustrated here using purified RdRps from hepatitis C virus and foot-and-mouth disease virus. The potential of fluorescence-based approaches for the enzymatic characterization of viral polymerases, as well as for high-throughput screening of antiviral drugs, are discussed.
Zika 病毒（ZIKV）是一种新兴病原体，与大量严重神经疾病有关，包括格林-巴利综合征和小头症。尽管它最近被确定为一个严重的全球公共卫生问题，但没有许可的治疗方法来控制这种病毒。因此，迫切需要开发高通量筛选抗病毒药物的方法。我们在这里描述了一种基于荧光的方法来监测 Zika 病毒 RNA 依赖性 RNA 聚合酶（RdRp）的实时聚合活性。通过使用均聚物 RNA 模板分子，可以用荧光染料检测从头合成 RNA，从而可以特异性定量和测定双链 RNA 的形成动力学。使用这种基于荧光的测定法检测到的 ZIKV RdRp 活性与测量放射性标记核苷酸掺入的传统测定法呈正相关。我们还通过使用已知的广谱抑制剂鉴定针对病毒聚合酶的 ZIKV 抑制剂来验证了这种方法作为合适的测定法。该测定法还成功地适应于检测不同 RdRps 的 RNA 聚合酶活性，这里通过丙型肝炎病毒和口蹄疫病毒的纯化 RdRps 进行了说明。讨论了荧光法在病毒聚合酶的酶学表征以及抗病毒药物的高通量筛选中的潜在应用。
