Abstract
The rapid mutation of the SARS-CoV-2 spike (S) protein promotes immune evasion and challenges current therapeutics. However, its cysteine/disulfide bond pattern is highly conserved across coronaviruses and essential for its structure and function. Here, we demonstrate that the folding and maturation of the S protein is a redox-sensitive process managed by sulfhydryl oxidase Ero1α and protein disulfide isomerase (PDI) in the endoplasmic reticulum. Reducing agents or cysteine mutations inhibit furin-processing of the S protein and its transport to the plasma membrane. Genetic or pharmacological disruption of the Ero1α-PDI pathway impairs S protein oxidative folding, furin cleavage and plasma membrane localization, as well as its fusogenic activity. Using a SARS-CoV-2 reverse genetics model, we confirm that targeting the Ero1α-PDI pathway exhibits potent antiviral effect. Moreover, the Ero1α-PDI pathway is essential for S protein maturation across different β-coronaviruses. Thus, our findings highlight the oxidative protein folding machinery as a promising target for developing broad-spectrum anti-coronavirus therapies.