Tan Hantai, Zhang Senzhao, Wu Zhen, He Yu, Wang Tao, Tan Wangyang, Tang Xuedan, Li Wei, Wang Mingshu, Jia Renyong, Zhu Dekang, Liu Mafeng, Zhao Xinxin, Yang Qiao, Wu Ying, Zhang Shaqiu, Huang Juan, Ou Xumin, Sun Di, Tian Bin, Cheng Anchun, Chen Shun
Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Engineering Research Center of Southwest Animal Disease Prevention and Control Technology, Ministry of Education of the People's Republic of China, Chengdu 611130, China.
Poult Sci. 2024 Dec;103(12):104322. doi: 10.1016/j.psj.2024.104322. Epub 2024 Sep 12.
Duck Tembusu virus (DTMUV) of the Orthoflavivirus genus poses a significant threat to waterfowl aquaculture. Nonstructural protein 1 (NS1), a multifunctional glycoprotein, exists in various oligomeric forms and performs diverse functions. The greasy finger (GF) region within NS1 of other flaviviruses has been shown to be a crucial component of the hydrophobic protrusion aiding in anchoring NS1 to the endoplasmic reticulum (ER). However, detailed studies on the role of the GF region in viral proliferation in vitro and the biological properties of NS1 remain scarce. A series of recombinant DTMUV (rDTMUV) with mutations in the GF region, including NS1-F158A, G159A, F160A, G161A, V162A, L163A, F160R, multipoint mutations (GF-4M), or regional deletions (ΔGF), were rescued using a DNA-based reverse genetics system. Only 5 rDTMUV variants (G159A, F160A, G161A, V162A, and L163A) could be rescued successfully, and these mutations were found to impair replication, reduce virulence, and decrease plaque size, as shown by growth kinetics, duck embryo virulence, and plaque assays, respectively. Upon examining NS1 expression by western blot, we discovered that secreted NS1 (sNS1) presented in large quantities in the supernatant of cells infected with rDTMUV-NS1-G159A, whereas intracellular NS1 was less abundant. These mutations also impacted the primary forms and secretion rates of NS1 in cases of overexpression by western blot and indirect ELISA. Exception for F160A and G161A, which showed decreased secretion rates, all other mutations increased sNS1 expression, with the most pronounced increase observed in F158A and ΔGF, and rDTMUV with these mutations can't be rescued. Co-localization studies of NS1 with the ER demonstrated that the ΔGF mutation attenuated NS1 anchoring to the ER, thereby inhibiting its intracellular residence and promoting secretion. Although these effects vary between flaviviruses, our data reveal that the GF region of NS1 is crucial for viral proliferation and NS1 secretion.
黄病毒属的鸭坦布苏病毒（DTMUV）对水禽养殖业构成重大威胁。非结构蛋白1（NS1）是一种多功能糖蛋白，以多种寡聚形式存在并发挥多种功能。其他黄病毒NS1内的油腻指（GF）区域已被证明是疏水突起的关键组成部分，有助于将NS1锚定在内质网（ER）上。然而，关于GF区域在体外病毒增殖中的作用以及NS1生物学特性的详细研究仍然很少。使用基于DNA的反向遗传学系统拯救了一系列GF区域发生突变的重组DTMUV（rDTMUV），包括NS1-F158A、G159A、F160A、G161A、V162A、L163A、F160R、多点突变（GF-4M）或区域缺失（ΔGF）。只有5种rDTMUV变体（G159A、F160A、G161A、V162A和L163A）能够成功拯救，并且这些突变分别通过生长动力学、鸭胚毒力和蚀斑试验表明损害复制、降低毒力并减小蚀斑大小。通过蛋白质印迹检测NS1表达时，我们发现分泌型NS1（sNS1）在感染rDTMUV-NS1-G159A的细胞上清液中大量存在，而细胞内NS1含量较少。这些突变在蛋白质印迹和间接ELISA过表达的情况下也影响NS1的主要形式和分泌率。除了F160A和G161A分泌率降低外，所有其他突变均增加了sNS1表达，在F158A和ΔGF中观察到最明显的增加，并且具有这些突变的rDTMUV无法拯救。NS1与ER的共定位研究表明，ΔGF突变减弱了NS1锚定到ER，从而抑制其在细胞内的停留并促进分泌。尽管这些影响在黄病毒之间有所不同，但我们的数据表明NS1的GF区域对于病毒增殖和NS1分泌至关重要。
