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.

中文翻译：

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阻断N-和O-糖精制后对SARS-CoV-2病毒进入体外的抑制作用

SARS-CoV-2的Spike蛋白，其受体结合域（RBD）及其主要受体ACE2被广泛糖基化。该翻译后修饰对病毒进入的影响尚未确定。我们在CRISPR-Cas9糖工程化细胞中表达了Spike蛋白和ACE2的不同糖型，并开发了相应的SARS-CoV-2伪病毒。我们观察到N-和O-聚糖对Spike-ACE2结合的贡献很小。但是，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基夫农碱在寡甘露糖阶段阻断N-聚糖的生物合成，可显着减少病毒进入表达ACE2的HEK293T细胞。阻断O-聚糖的加工也部分阻断了病毒的进入。机理研究表明，病毒进入过程中聚糖具有多种作用。其中，抑制N-聚糖的生物合成可增强Spike蛋白的蛋白水解作用。这可以减少RBD在病毒上的表现，降低与宿主ACE2的结合并减少病毒进入。总体而言，可以评估糖基化的化学抑制剂的COVID-19。