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.