Abstract
Hydrogen peroxide (H(2)O(2)) exhibits broad-spectrum antiviral activity and is commonly used as an over-the-counter disinfecting agent. However, its potential activities against SARS-CoV-2 have not been systematically evaluated, and mechanisms of action are not well understood. In this study, we investigate H(2)O(2)'s antiviral activity against SARS-CoV-2 infection and its impact on the virion's structural integrity as compared to the commonly used fixative agent paraformaldehyde (PFA). We show that H(2)O(2) rapidly and directly inactivates SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 0.0015%. Cryogenic electron tomography (cryo-ET) with subtomogram averaging reveals that treatment with PFA induced the viral trimeric spike protein (S) to adopt a post-fusion conformation, and treatment of viral particles with H(2)O(2) locked S in its pre-fusion conformation. Therefore, H(2)O(2) treatment likely has induced modifications, such as oxidation of cysteine residues within the S subunits of the spike trimer that locked them in their pre-fusion conformation. Locking of the meta-stable pre-fusion trimer prevents its transition to the post-fusion conformation, a process essential for viral fusion with host cells and entry into host cells. Together, our cellular, biochemical, and structural studies established that hydrogen peroxide can inactivate SARS-CoV-2 in tissue culture and uncovered its underlying molecular mechanism.IMPORTANCEHydrogen peroxide (H(2)O(2)) is the commonly used, over-the-counter antiseptic solution available in pharmacies, but its effect against the SARS-CoV-2 virus has not been evaluated systematically. In this study, we show that H(2)O(2) inactivates the SARS-CoV-2 infectivity and establish the effective concentration of this activity. Cryogenic electron tomography and sub-tomogram averaging reveal a detailed structural understanding of how H(2)O(2) affects the SARS-CoV-2 spike in comparison with that of the commonly used fixative PFA under identical conditions. We found that PFA promoted a post-fusion conformation of the viral spike protein, while H(2)O(2) could potentially lock the spike in its pre-fusion state. Our findings not only substantiate the disinfectant efficacy of H(2)O(2) as a potent agent against SARS-CoV-2 but also lay the groundwork for future investigations into targeted antiviral therapies that may leverage the virus' structural susceptibilities. In addition, this study may have significant implications for developing new antiviral strategies and improving existing disinfection protocols.