Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Průmyslová 595, 25242, Vestec, Czech Republic.
Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Belgium.
J Microbiol Immunol Infect. 2022 Apr;55(2):191-198. doi: 10.1016/j.jmii.2021.08.008. Epub 2021 Aug 25.
Trichomonas vaginalis is the causative agent of a sexually transmitted disease in humans. The virulence of the parasite depends on multiple factors, including the presence of endosymbiotic dsRNA viruses. The presence of Trichomonasviruses (TVV) was associated with more severe genital symptoms, increased proinflammatory host reactions, and modulated parasite sensitivity to metronidazole. However, no efficient antiviral drugs are available against TVV to derive isogenic TVV-positive and TVV-negative cell lines that are essential for investigations of the TVV impact on T. vaginalis biology.
7-Deaza-2'-C-methyladenosine (7d2CMA) and 2'-C-methylcytidine (2CMC) were used for TVV inhibitory assay. TVV replication was monitored using quantitative reverse transcription PCR (RT qPCR) and western blotting. Modeling of TVV1 RNA-dependent RNA polymerase (RdRp) was performed to visualize the inhibitor-RdRp interaction. Susceptibility to metronidazole was performed under aerobic and anaerobic conditions.
We demonstrated that 2CMC but not 7d2CMA is a potent inhibitor of TVV replication. Molecular modeling suggested that the RdRp active site can accommodate 2CMC in the active triphosphate nucleotide form. The effect of 2CMC was shown on strains infected with a single and multiple TVV species. The optimal 2CMC concentration (10 μM) demonstrated strong selectivity for TVVs over trichomonad growth. The presence of TVV has no effect on T. vaginalis metronidazole susceptibility in derived isogenic cell lines.
2CMC acts against TVVs and represents a new inhibitor against Totiviridae viruses. Our isogenic clones are now available for further studies of various aspects of T. vaginalis biology related to TVV infection.
阴道毛滴虫是一种人类性传播疾病的病原体。寄生虫的毒力取决于多个因素，包括内共生双链 RNA 病毒的存在。阴道毛滴虫病毒（TVV）的存在与更严重的生殖器症状、增加的促炎宿主反应以及调节寄生虫对甲硝唑的敏感性有关。然而，目前没有针对 TVV 的有效抗病毒药物，无法获得 TVV 阳性和 TVV 阴性的同源细胞系，而这些细胞系对于研究 TVV 对阴道毛滴虫生物学的影响至关重要。
使用 7-脱氮-2'-C-甲基腺苷（7d2CMA）和 2'-C-甲基胞苷（2CMC）进行 TVV 抑制测定。使用定量逆转录 PCR（RT qPCR）和 Western blot 监测 TVV 复制。进行 TVV1 RNA 依赖性 RNA 聚合酶（RdRp）建模，以可视化抑制剂-RdRp 相互作用。在需氧和厌氧条件下进行甲硝唑敏感性测定。
我们证明 2CMC 但不是 7d2CMA 是 TVV 复制的有效抑制剂。分子建模表明 RdRp 活性位点可以容纳 RdRp 活性三磷酸核苷酸形式的 2CMC。2CMC 的作用在感染单一和多种 TVV 物种的菌株中得到了证实。最佳 2CMC 浓度（10 μM）对 TVV 具有很强的选择性，而对阴道毛滴虫生长的选择性较低。在衍生的同源细胞系中，TVV 的存在对阴道毛滴虫甲硝唑敏感性没有影响。
2CMC 对 TVV 有效，代表了一种针对 Totiviridae 病毒的新型抑制剂。我们的同源克隆现在可用于进一步研究与 TVV 感染相关的各种阴道毛滴虫生物学方面。
