Abstract

The spike (S) protein of SARS-CoV-2 is extensively glycosylated, with N-glycosylation sites remaining highly conserved during viral evolution. While inhibiting N-glycosylation has been shown to significantly suppress SARS-CoV-2 infection, the underlying molecular mechanisms remain incompletely characterized. Here, we identify that two N-glycosylation sites, N61 and N343, are critical for spike maturation. We demonstrate that asparagine-to-aspartic acid substitutions (N to D) at these sites lead to endoplasmic reticulum (ER) retention of the S protein, with consequent abrogation of S1/S2 cleavage and near-complete elimination of viral infectivity. IP-MS analysis further reveals that the COPI complex, which facilitates retrograde Golgi-to-ER transport, is a key participant in this ER retention process. Additionally, inhibition of COPI effectively restores the plasma membrane localization of N61D- and N343D-mutated S proteins and enhanced viral infectivity. More importantly, a specific inhibitor has been developed that effectively blocks the ER-to-Golgi trafficking of the S protein, thereby broadly abolishing viral infectivity across SARS-CoV-2 variants. Overall, our study reveals the unique roles of N-glycosylation in the regulation of S protein maturation, providing a potential mechanistic target for antiviral drug development.