Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province 611130, China.
Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province 611130, China; Institute of Preventive Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China.
Vet Microbiol. 2022 Jun;269:109433. doi: 10.1016/j.vetmic.2022.109433. Epub 2022 Apr 25.
Flavivirus nonstructural protein 5 (NS5) harbors the N-terminal methyltransferase (MTase) and C-terminal polymerase RNA-dependent RNA polymerase (RdRp). The intramolecular NS5 features an integral MTase and RdRp interface with two components: a six-residue hydrophobic network and a GTR linker. Herein, the determinants of the MTase-RdRp interface and flavivirus substituted GTR linker were explored in TMUV replication and proliferation. First, the NanoLuc® Binary Technology (NanoBiT) and coimmunoprecipitation assays (Co-IP) methods confirmed the interaction between the MTase and RdRp domains of TMUV NS5. To screen for an optimal orientation for reporter gene fusion to the protein of interest, the signal activity of eight combinations of MTase and RdRp was explored. Intriguingly, all the combinations with the reporter gene fused to the C-terminal of MTase (1.1 C/2.1 C MTase) could barely detect any positive signal, suggesting a role for the GTR linker of the MTase C-terminal in MTase-RdRp affinity. Based on the flavivirus NS5 homologous interplay, we introduced alanine mutations into the MTase-RdRp interface of TMUV NS5. However, no single or pairwise mutation was found to abort the NS5 intramolecular interaction. Then, a mutated replicon and infectious clone were constructed to analyze the replication ability and properties of the recombinant virus. The mutant replicons of MTase F113A and M115A replicated to comparable extent as the wild type (WT). However, the replication level of the mutant MTase W121A was impaired without an obvious decrease in proliferation and virulence. Both the RdRp F351A and P585A mutants could replicate and proliferate well. Notably, the RdRp F467A virus was attenuated and did not strikingly impair the MTase-RdRp interaction. Furthermore, the TMUV was specifically compatible with the substituted NS5 with a Japanese encephalitis virus (JEV) GTR linker. Compensatory mutations were observed in the context of a defective MTase-RdRp interface after several passages of the rescued mutants in BHK-21 cells. A greater understanding of the molecular mechanism of the NS5 protein controlling duck TMUV replication will facilitate the design of novel therapies.
黄病毒非结构蛋白 5（NS5）具有 N 端甲基转移酶（MTase）和 C 端聚合酶 RNA 依赖性 RNA 聚合酶（RdRp）。NS5 分子内具有完整的 MTase 和 RdRp 界面，由两个组件组成：一个六残基疏水性网络和一个 GTR 接头。在此，通过 TMUV 复制和增殖研究了 MTase-RdRp 界面和黄病毒取代 GTR 接头的决定因素。首先，使用 NanoLuc®Binary Technology（NanoBiT）和免疫共沉淀（Co-IP）方法证实了 TMUV NS5 的 MTase 和 RdRp 结构域之间的相互作用。为了筛选报告基因融合到目标蛋白的最佳取向，探索了 MTase 和 RdRp 之间的八种组合的信号活性。有趣的是，所有与 C 端 MTase 融合报告基因的组合（1.1C/2.1C MTase）几乎检测不到任何阳性信号，这表明 MTase C 端 GTR 接头在 MTase-RdRp 亲和力中起作用。基于黄病毒 NS5 同源相互作用，我们在 TMUV NS5 的 MTase-RdRp 界面中引入了丙氨酸突变。然而，没有发现单个或成对突变会终止 NS5 分子内相互作用。然后，构建了突变复制子和感染性克隆以分析重组病毒的复制能力和特性。MTase F113A 和 M115A 的突变复制子复制到与野生型（WT）相当的程度。然而，MTase W121A 的突变复制水平受损，增殖和毒力没有明显下降。RdRp F351A 和 P585A 突变体均能良好复制和增殖。值得注意的是，RdRp F467A 病毒的毒力减弱，并且不会明显损害 MTase-RdRp 相互作用。此外，TMUV 与具有日本脑炎病毒（JEV）GTR 接头的替代 NS5 特别兼容。在 BHK-21 细胞中传代几次后，在有缺陷的 MTase-RdRp 界面背景下观察到补偿突变。对 NS5 蛋白控制鸭 TMUV 复制的分子机制有了更深入的了解，将有助于设计新的治疗方法。
