N-glycosylation plays an important role in the pathogenesis of viral infections. However, the role of SARS-CoV-2 RBD N-glycosylation in viral entry remains elusive. In this study, we expressed and purified N331 and N343 N-glycosite mutants of SARS-CoV-2 RBD. We found that de-glycosylation at N331 and N343 drastically reduces the RBD binding to ACE2. More importantly, based on qualitative and quantitative virology research methods, we show that the mutation of RBD N-glycosites interfered with SARS-CoV-2 internalization rather than attachment potentially by decreasing RBD binding to the receptors. Also, the double N-glycosites mutant (N331 + N343) showed significantly increased sensitivity against the designated RBD neutralizing antibodies. Taken together, these results suggest that N-glycosylation of SARS-CoV-2 RBD is not only critical for viral internalization into respiratory epithelial cells but also shields the virus from neutralization. It may provide new insights into the biological process of early-stage SARS-CoV-2 infection with potential therapeutic implications.

N-糖基化在病毒感染的发病机制中起重要作用。然而，SARS-CoV-2 RBD N-糖基化在病毒进入中的作用仍然难以捉摸。在这项研究中，我们表达并纯化了 SARS-CoV-2 RBD 的 N331 和 N343 N 糖位点突变体。我们发现 N331 和 N343 的去糖基化显着降低了 RBD 与 ACE2 的结合。更重要的是，基于定性和定量病毒学研究方法，我们表明 RBD N-糖位点的突变干扰了 SARS-CoV-2 内化，而不是通过降低 RBD 与受体的结合来潜在地附着。此外，双 N 糖位点突变体 (N331 + N343) 对指定的 RBD 中和抗体的敏感性显着增加。综合起来，这些结果表明 SARS-CoV-2 RBD 的 N-糖基化不仅对病毒内化进入呼吸道上皮细胞至关重要，而且还可以保护病毒免受中和。它可能为具有潜在治疗意义的早期 SARS-CoV-2 感染的生物学过程提供新的见解。