Department of Food Sciences and Nutrition, Mukogawa Women's University, 6-46 Ikebiraki, Nishinomiya, Hyogo, 663-8558, Japan.
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka, 563-8577, Japan.
Virol J. 2021 Aug 28;18(1):177. doi: 10.1186/s12985-021-01644-7.
The development of an influenza RNA-dependent RNA polymerase (RdRp) inhibitor is required; therefore, a method for evaluating the activity of influenza RdRp needs to be developed. The current method uses an ultracentrifuge to separate viral particles and quantifies RdRp activity with radioisotope-labeled nucleosides, such as P-GTP. This method requires special equipment and radioisotope management, so it cannot be implemented in all institutions. We have developed a method to evaluate the mRNA transcription activity of RdRp without using ultracentrifugation and radioisotopes.
RdRp was extracted from viral particles that were purified from the culture supernatant using anionic polymer-coated magnetic beads that can concentrate influenza virus particles from the culture supernatant in approximately 30 min. A strand-specific real-time reverse transcription polymerase chain reaction (RT-PCR) method was developed based on reverse transcription using tagged primers. RT primers were designed to bind to a sequence near the 3' end of mRNA containing a poly A tail for specific recognition of the mRNA, with an 18-nucleotide tag attached to the 5' end of the sequence. The RT reaction was performed with this tagged RT primer, and the amount of mRNA was analyzed using real-time qPCR. Real-time qPCR using the tag sequence as the forward primer and a segment-specific reverse primer ensured the specificity for quantifying the mRNA of segments 1, 4, and 5. The temperature, reaction time, and Mg concentration were determined to select the optimum conditions for in vitro RNA synthesis by RdRp, and the amount of synthesized mRNAs of segments 1, 4, and 5 was determined with a detection sensitivity of 10 copies/reaction. In addition, mRNA synthesis was inhibited by ribavirin triphosphate, an RdRp inhibitor, thus indicating the usefulness of this evaluation method for screening RdRp inhibitors.
This method makes it possible to analyze the RdRp activity even in a laboratory where ultracentrifugation and radioisotopes cannot be used. This novel method for measuring influenza virus polymerase activity will further promote research to identify compounds that inhibit viral mRNA transcription activity of RdRp.
需要开发一种流感 RNA 依赖性 RNA 聚合酶（RdRp）抑制剂，因此需要开发一种评估流感 RdRp 活性的方法。目前的方法使用超速离心分离病毒颗粒，并使用放射性同位素标记的核苷（如 P-GTP）定量 RdRp 活性。该方法需要特殊的设备和放射性同位素管理，因此不能在所有机构实施。我们开发了一种无需超速离心和放射性同位素即可评估 RdRp mRNA 转录活性的方法。
使用带负电荷的聚合物包被的磁性珠从培养上清液中纯化的病毒颗粒中提取 RdRp，带负电荷的聚合物包被的磁性珠可在大约 30 分钟内从培养上清液中浓缩流感病毒颗粒。基于标记引物的逆转录，开发了一种用于 RdRp 的链特异性实时逆转录聚合酶链反应（RT-PCR）方法。RT 引物设计为与含有多 A 尾巴的 mRNA 3' 端附近的序列结合，以特异性识别 mRNA，并在序列的 5' 端添加 18 个核苷酸的标签。使用该标记 RT 引物进行 RT 反应，并使用实时 qPCR 分析 mRNA 的量。使用标签序列作为正向引物和特定片段的反向引物进行实时 qPCR 可确保定量分析片段 1、4 和 5 的 mRNA 的特异性。确定了温度、反应时间和 Mg 浓度，以选择 RdRp 体外 RNA 合成的最佳条件，并使用 10 个拷贝/反应的检测灵敏度确定了片段 1、4 和 5 的合成 mRNA 的量。此外，RdRp 抑制剂利巴韦林三磷酸抑制了 mRNA 的合成，表明该评估方法在筛选 RdRp 抑制剂方面是有用的。
即使在不能使用超速离心和放射性同位素的实验室中，该方法也可以分析 RdRp 活性。这种测量流感病毒聚合酶活性的新方法将进一步促进识别抑制 RdRp 介导的病毒 mRNA 转录活性的化合物的研究。
