Disruption of spike protein N-glycosylation induces its endoplasmic reticulum retention and attenuates SARS-CoV-2 infectivity.

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.IMPORTANCEN-glycosylation of the spike protein is critical for SARS-CoV-2. While most studies have focused on the effects on spike-ACE2 binding and neutralizing antibody recognition, few studies have reported how N-glycosylation regulates S protein maturation, with the underlying molecular mechanisms remaining poorly understood. Here, we demonstrate that N-glycosylation at N61/ N343 contributes to spike ER-to-Golgi trafficking. Specifically, defects in S protein's N-glycosylation (including mutations at N61 or N343, N-glycosylation inhibitors treatment, and MOGS depletion) result in ER retention through COPI-mediated retrograde Golgi-to-ER transport, and thus, the S proteins are not effectively cleaved by furin in the Golgi. This impairment of S protein maturation leads to a significant reduction in viral infectivity, which highlights the key role of N-glycosylation at residues N61 and N343 in SARS-CoV-2 life cycle. Overall, our findings uncover the molecular mechanism by which N-glycosylation controls SARS-CoV-2 spike intracellular trafficking, offering novel insights for anti-SARS-CoV-2 strategies.