Palmitoylation of CLDN12 regulated by ZDHHC7 and APT1/2 promotes hepatitis C virus entry

Hepatitis C virus (HCV) entry is a critical step in establishing infection and is highly dependent on host cell co-receptors. We previously identified that the tight junction protein CLDN12 facilitates HCV entry. However, the regulatory mechanisms governing the dynamic post-translational modifications of CLDN12 remain unclear. This study employs targeted mutagenesis, palmitoylation level analysis, plasma membrane localization, manipulation of key enzymes, and functional virology assays to reveal whether and how reversible palmitoylation regulates the function of CLDN12 in HCV entry. We found that the palmitoylation occurs at cysteine residues C3 and C109 of CLDN12, a modification critical for its role in mediating HCV entry. The palmitoyltransferase ZDHHC7 is a key enzyme that catalyzes the palmitoylation of CLDN12, positively promoting HCV entry. Acyl-protein thioesterases APT1 and APT2 regulate the de-palmitoylation of CLDN12 through differential modulation at the two sites, thereby negatively regulating HCV entry. Our study discovers that ZDHHC7 and APT1/2 form a reversible palmitoylation switch, which regulates the efficiency of HCV entry by dynamically controlling the plasma membrane localization of CLDN12. This finding reveals a new mechanism by which HCV exploits the host's palmitoylation cycle to promote infection, and highlights this regulatory axis as a potential target for antiviral intervention. IMPORTANCE: HCV infection poses a significant global health burden. Despite the remarkable efficacy of direct antiviral agents, developing new strategies targeting host factors is crucial for addressing drug resistance and re-infection. Viral entry is a key step in establishing infection, where the host factor CLDN12 promotes HCV invasion. However, the regulatory mechanisms controlling the plasma membrane localization of CLDN12 remain unclear. This study elucidates the mechanism of CLDN12 palmitoylation, regulated by the palmitoyltransferase ZDHHC7 and the acyl-protein thioesterases APT1 and APT2. This dynamic palmitoylation switch governs the plasma membrane localization and function of CLDN12 through reversible modifications, thereby regulating the efficiency of HCV entry. This discovery not only expands our understanding of the network of host palmitoylation regulatory mechanisms involved in HCV entry but also reveals a finely tuned dynamic equilibrium model characterized by the collaborative regulation of palmitoyltransferases and acyl-protein thioesterases, with a preference for specific sites.