Institute of Fundamental Medicine and Biology, Kazan Federal University, Kremlyovskaya Street 18, 420008, Kazan, Russia.
Institute of Medical Virology, Justus Liebig University, Schubertstrasse 81, 35392, Giessen, Germany.
Virol J. 2018 Jan 5;15(1):5. doi: 10.1186/s12985-017-0915-1.
Influenza is a severe contagious disease especially in children, elderly and immunocompromised patients. Beside vaccination, the discovery of new anti-viral agents represents an important strategy to encounter seasonal and pandemic influenza A virus (IAV) strains. The bacterial extra-cellular ribonuclease binase is a well-studied RNase from Bacillus pumilus. Treatment with binase was shown to improve survival of laboratory animals infected with different RNA viruses. Although binase reduced IAV titer in vitro and in vivo, the mode of action (MOA) of binase against IAV at the molecular level has yet not been studied in depth and remains elusive.
To analyze whether binase impairs virus replication by direct interaction with the viral particle we applied a hemagglutination inhibition assay and monitored the integrity of the viral RNA within the virus particle by RT-PCR. Furthermore, we used Western blot and confocal microscopy analysis to study whether binase can internalize into MDCK-II cells. By primer extension we examined the effect of binase on the integrity of viral RNAs within the cells and using a mini-genome system we explored the effect of binase on the viral expression.
We show that (i) binase does not to attack IAV particle-protected viral RNA, (ii) internalized binase could be detected within the cytosol of MDCK-II cells and that (iii) binase impairs IAV replication by specifically degrading viral RNA species within the infected MDCK-II cells without obvious effect on cellular mRNAs.
Our data provide novel evidence suggesting that binase is a potential anti-viral agent with specific intra-cellular MOA.
流感是一种严重的传染病，尤其在儿童、老年人和免疫功能低下的患者中更为常见。除了接种疫苗外，发现新的抗病毒药物是应对季节性和大流行性甲型流感病毒（IAV）株的重要策略。细菌胞外核糖核酸酶 binase 是一种来自枯草芽孢杆菌的研究充分的 RNase。研究表明，binase 治疗可提高感染不同 RNA 病毒的实验动物的存活率。尽管 binase 降低了 IAV 的体外和体内滴度，但 binase 对 IAV 的作用机制（MOA）在分子水平上尚未得到深入研究，仍不清楚。
为了分析 binase 是否通过与病毒颗粒的直接相互作用来损害病毒复制，我们应用血凝抑制试验并通过 RT-PCR 监测病毒颗粒内病毒 RNA 的完整性。此外，我们使用 Western blot 和共聚焦显微镜分析来研究 binase 是否可以内化到 MDCK-II 细胞中。通过引物延伸，我们检查了 binase 对细胞内病毒 RNA 完整性的影响，并且使用迷你基因组系统探索了 binase 对病毒表达的影响。
我们表明：（i）binase 不会攻击 IAV 颗粒保护的病毒 RNA；（ii）在 MDCK-II 细胞的细胞质中可以检测到内化的 binase；（iii）binase 通过特异性降解感染的 MDCK-II 细胞内的病毒 RNA 种类来损害 IAV 复制，而对细胞内 mRNAs 没有明显影响。
我们的数据提供了新的证据，表明 binase 是一种具有特定细胞内 MOA 的潜在抗病毒药物。
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