Chemical & Biological Engineering, State University of New York, Buffalo, United States.
Biomedical Engineering, State University of New York, Buffalo, United States.
Elife. 2020 Oct 26;9:e61552. doi: 10.7554/eLife.61552.
The Spike protein of SARS-CoV-2, its receptor-binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding. However, these carbohydrates played a major role in regulating viral entry. Blocking N-glycan biosynthesis at the oligomannose stage using both genetic approaches and the small molecule kifunensine dramatically reduced viral entry into ACE2 expressing HEK293T cells. Blocking O-glycan elaboration also partially blocked viral entry. Mechanistic studies suggest multiple roles for glycans during viral entry. Among them, inhibition of N-glycan biosynthesis enhanced Spike-protein proteolysis. This could reduce RBD presentation on virus, lowering binding to host ACE2 and decreasing viral entry. Overall, chemical inhibitors of glycosylation may be evaluated for COVID-19.
SARS-CoV-2 的刺突蛋白、其受体结合域 (RBD) 和主要受体 ACE2 广泛糖基化。这种翻译后修饰对病毒进入的影响尚未确定。我们在 CRISPR-Cas9 糖基化工程细胞中表达了 Spike 蛋白和 ACE2 的不同糖型，并开发了相应的 SARS-CoV-2 假病毒。我们观察到 N- 和 O-聚糖对 Spike-ACE2 结合的贡献很小。然而，这些碳水化合物在调节病毒进入中起主要作用。使用遗传方法和小分子 kifunensine 阻断寡甘露糖阶段的 N-聚糖生物合成，可显著降低 ACE2 表达的 HEK293T 细胞中的病毒进入。阻断 O-聚糖的延伸也部分阻断了病毒进入。机制研究表明，聚糖在病毒进入过程中具有多种作用。其中，抑制 N-聚糖生物合成增强了 Spike 蛋白的蛋白水解。这可能会降低病毒表面 RBD 的呈现，降低与宿主 ACE2 的结合并减少病毒进入。总之，糖基化的化学抑制剂可能会针对 COVID-19 进行评估。
