State Key Laboratory of Common Mechanism Research for Major Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; and School of Basic Medicine, Peking Union Medical College, Beijing, 100005, China.
Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; and School of Basic Medicine, Peking Union Medical College, Beijing, 100005, China.
J Biomed Sci. 2024 Jun 7;31(1):60. doi: 10.1186/s12929-024-01048-z.
Flavivirus is a challenge all over the world. The replication of flavivirus takes place within membranous replication compartments (RCs) derived from endoplasmic reticulum (ER). Flavivirus NS1 proteins have been proven essential for the formation of viral RCs by remodeling the ER. The glycosylation of flavivirus NS1 proteins is important for viral replication, yet the underlying mechanism remains unclear.
HeLa cells were used to visualize the ER remodeling effects induced by NS1 expression. ZIKV replicon luciferase assay was performed with BHK-21 cells. rZIKV was generated from BHK-21 cells and the plaque assay was done with Vero Cells. Liposome co-floating assay was performed with purified NS1 proteins from 293T cells.
We found that the glycosylation of flavivirus NS1 contributes to its ER remodeling activity. Glycosylation deficiency of NS1, either through N-glycosylation sites mutations or tunicamycin treatment, compromises its ER remodeling activity and interferes with viral RCs formation. Disruption of NS1 glycosylation results in abnormal aggregation of NS1, rather than reducing its membrane-binding activity. Consequently, deficiency in NS1 glycosylation impairs virus replication.
In summary, our results highlight the significance of NS1 glycosylation in flavivirus replication and elucidate the underlying mechanism. This provides a new strategy for combating flavivirus infections.
黄病毒在全球范围内都是一个挑战。黄病毒的复制发生在内质网（ER）衍生的膜复制隔间（RC）中。黄病毒 NS1 蛋白已被证明通过重塑 ER 对于病毒 RC 的形成是必不可少的。黄病毒 NS1 蛋白的糖基化对于病毒复制很重要，但潜在的机制尚不清楚。
使用 HeLa 细胞观察 NS1 表达诱导的 ER 重塑效应。使用 BHK-21 细胞进行 ZIKV 复制子荧光素酶测定。用 BHK-21 细胞生成 rZIKV，并用 Vero 细胞进行噬斑测定。用来自 293T 细胞的纯化 NS1 蛋白进行脂质体共浮实验。
我们发现黄病毒 NS1 的糖基化有助于其 ER 重塑活性。通过 N-糖基化位点突变或衣霉素处理使 NS1 糖基化缺乏，会损害其 ER 重塑活性并干扰病毒 RC 的形成。NS1 糖基化的破坏导致 NS1 的异常聚集，而不是降低其膜结合活性。因此，NS1 糖基化的缺乏会损害病毒复制。
总之，我们的结果强调了 NS1 糖基化在黄病毒复制中的重要性，并阐明了潜在的机制。这为防治黄病毒感染提供了新的策略。
