Identification of Prohibitin (PHB) as an Entry Factor of Highly Pathogenic Coronaviruses Using Metabolic Glycoengineering and Photo-Crosslinking Techniques.

Coronaviruses (CoVs) rely on host surface factors to mediate viral attachment and entry, yet conserved host cofactors shared across different CoVs remain poorly defined. Here, we established a host-factor capture platform based on metabolic glycoengineering and photo-crosslinking to identify host factors associated with CoV Spike proteins under near-physiological conditions. Using this approach, we identified prohibitin (PHB) as a candidate Spike-associated host factor shared by SARS-CoV, MERS-CoV, and SARS-CoV-2. Functional analyses showed that depletion of membrane-associated PHB decreased pseudovirus infection, whereas PHB reconstitution or overexpression enhanced susceptibility across multiple cell systems, including Huh7, BEAS-2B, Calu-3, and primary human airway epithelial cells (hAECs). In addition, antibody-mediated blockade of cell-surface PHB suppressed pseudovirus entry in both hAECs and human lung organoids. Immunohistochemical analyses further demonstrated PHB expression in normal human lung tissues. Mechanistically, recombinant PHB interacted with the Spike proteins of all three CoVs, and this association was supported by surface plasmon resonance and co-immunoprecipitation assays. Structural prediction combined with fragment-based alanine scanning identified residues 139-154 within the extracellular PHB domain as a critical region for efficient Spike association. Furthermore, PHB-associated pseudovirus entry was sensitive to lipid raft disruption, supporting a role for cholesterol-dependent membrane microdomains in this process. Together, these findings identify membrane-associated PHB as a conserved Spike-associated host factor linked to CoV entry.