ZDHHC18‐Mediated Palmitoylation of ORF3a Promotes SARS‐CoV‐2 Pathogenesis by Antagonizing TRIM16‐Mediated Ubiquitination and Proteasomal Degradation

SARS‐CoV‐2 accessory protein ORF3a contributes to viral pathogenesis through membrane remodeling, immune evasion, and inflammation induction. However, the molecular mechanisms underlying ORF3a‐mediated pathogenesis remain poorly characterized, and no therapeutic strategies targeting ORF3a currently exist. Here, we demonstrate that palmitoylation, a post‐translational modification, governs ORF3a‐mediated viral pathogenesis. Specifically, ORF3a undergoes ZDHHC18‐mediated palmitoylation at evolutionarily conserved Cys130/Cys133 residues, which stabilizes the protein by masking an intrinsic proteasomal degradation signal. This palmitoylation competitively inhibits tripartite motif‐containing 16 (TRIM16)‐dependent K27‐linked polyubiquitination, thereby preventing ORF3a degradation and enhancing viral replication and inflammatory responses. A designed ORF3a‐mimicking palmitoylation‐inhibitory peptide (OPIP) blocked ORF3a palmitoylation, promoted its degradation, and significantly reduced SARS‐CoV‐2 pathogenicity. Collectively, these findings establish ZDHHC18‐mediated palmitoylation as a central regulator of ORF3a stability and virulence, revealing a potentially druggable axis for disrupting SARS‐CoV‐2 pathogenesis.