Duck Tembusu virus (TMUV) belongs to the flavivirus genus whose genome replication involved in capping and RNA synthesis dominating by nonstructural protein 5 (NS5). Flaviviral replication has been well documented to occur in the cytoplasm, but the effect of NS5 to gain access to the nucleus remains controversial. Here, TMUV NS5 was observed to localize within the cytoplasm of transfected and infected cells and co-localized with the endoplasmic reticulum. We introduced two arginine mutations into the N390 and Q392 (N390R and Q392R) of the NS5 bipartite nuclear localization sequence (α/βNLS) and designated that mutagenesis as NS5_NLSmut, which has shown the ability to access the nucleus and hence attenuates viral replication and production in vitro. Additionally, there was no significant difference between the recovered wild-type TMUV (rTMUV-WT) and engineered mutant (rTMUV-NS5_NLSmut) on plaque morphology, survival rate of infected duck embryos or virus copies in tissues. Considering that NS5_NLSmut is mainly located in the cytoplasm of rTMUV-NS5_NLSmut infected cells at the early stage of infection. We further confirmed that NS5_NLSmut attenuated its interaction with nonstructural NS2B-NS3 (NS2B3) following transfection and infection. Meanwhile, the rTMUV-NS5_NLSmut tended to stimulate more interferon beta (IFNβ) than rTMUV-WT. However, preliminary study on transient NS5 and NS5_NLSmut detected the same levels of IFNβ mRNA mediated by RIG-I detection of NS5 RNA polymerase activity in cell. In summary, these results provide further insights into the relationship between the viral property and subcellular localization of flavivirus NS5 in terms of the NS5-NS2B3 interaction.

鸭坦布苏病毒 (TMUV) 属于黄病毒属，其基因组复制涉及由非结构蛋白 5 (NS5) 主导的加帽和 RNA 合成。黄病毒复制已被充分证明发生在细胞质中，但 NS5 进入细胞核的作用仍然存在争议。在这里，观察到 TMUV NS5 定位于转染和感染细胞的细胞质内，并与内质网共定位。我们在 NS5 二分核定位序列 (α/βNLS) 的 N390 和 Q392（N390R 和 Q392R）中引入了两个精氨酸突变，并将该突变指定为 NS5 _NLSmut，它已显示出进入细胞核的能力，从而减弱病毒复制和体外生产。此外，回收的野生型 TMUV (rTMUV-WT) 和工程突变体 (rTMUV-NS5 _NLSmut ) 在斑块形态、感染鸭胚胎的存活率或组织中的病毒拷贝方面没有显着差异。考虑到NS5 _NLSmut在感染早期主要位于rTMUV-NS5 _NLSmut感染细胞的细胞质中。我们进一步证实 NS5 _NLSmut在转染和感染后减弱了其与非结构性 NS2B-NS3 (NS2B3) 的相互作用。同时，与rTMUV-WT 相比，rTMUV-NS5 _NLSmut倾向于刺激更多的干扰素β（IFNβ）。然而，对瞬态 NS5 和 NS5 _{NLSmut 的}初步研究检测到由 RIG-I 检测细胞中 NS5 RNA 聚合酶活性介导的相同水平的 IFNβ mRNA。总而言之，这些结果提供了对 NS5-NS2B3 相互作用方面黄病毒 NS5 的病毒特性与亚细胞定位之间关系的进一步见解。