Loutan Arlo J, Yang Baiuyan, Connolly Gabrielle, Montoya Adam, Smiley Robert J, Chatterjee Arnab K, Götte Matthias
Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2E1, Canada.
Calibr-Skaggs Institute for Innovation Medicines at Scripps Research, La Jolla, CA 92037, USA.
Viruses. 2025 Mar 14;17(3):420. doi: 10.3390/v17030420.
The order includes a range of zoonotic viruses, which can cause severe disease in humans. The viral replication machinery is a logical target for the development of direct-acting antivirals. Inhibition of the cap-snatching endonuclease activity of related influenza viruses provides a proof of concept. Using the influenza B virus (IBV) RNA-dependent RNA polymerase complex as a benchmark, we conducted a comparative analysis of endonuclease activities of recombinant full-length bunyaviral L proteins using gel-based assays. The IBV complex demonstrates specific endonucleolytic cleavage and a clear preference for capped substrates. In contrast, severe fever with thrombocytopenia syndrome, Sin Nombre, and Hantaan virus L proteins readily cleave capped and uncapped RNAs to a broader spectrum of RNA fragments. Active site mutants further help to control for the potential of contaminating nucleases, exonuclease activity, and RNA hydrolysis. The influenza cap-snatching inhibitor baloxavir and derivatives have been used to validate this approach. In conclusion, the results of this study demonstrate the importance of assays with single nucleotide resolution and the use of full-length L proteins as a valuable experimental tool to identify selective endonuclease inhibitors.
该订单包含一系列人畜共患病毒，这些病毒可导致人类患上严重疾病。病毒复制机制是开发直接作用抗病毒药物的合理靶点。抑制相关流感病毒的帽状结构抢夺内切核酸酶活性提供了一个概念验证。以乙型流感病毒（IBV）的RNA依赖性RNA聚合酶复合物为基准，我们使用基于凝胶的测定法对重组全长布尼亚病毒L蛋白的内切核酸酶活性进行了比较分析。IBV复合物表现出特异性内切核酸酶切割以及对带帽底物的明显偏好。相比之下，严重发热伴血小板减少综合征病毒、辛诺柏病毒和汉坦病毒的L蛋白很容易将带帽和不带帽的RNA切割成更广泛的RNA片段谱。活性位点突变体进一步有助于控制潜在的核酸酶污染、核酸外切酶活性和RNA水解。流感帽状结构抢夺抑制剂巴洛沙韦及其衍生物已被用于验证这种方法。总之，本研究结果证明了单核苷酸分辨率测定以及使用全长L蛋白作为鉴定选择性内切核酸酶抑制剂的有价值实验工具的重要性。
