ZDHHC11-mediated palmitoylation of viral envelope protein restricts dengue virus replication: implication for the development of universal anti-flavivirus therapeutic strategies.

Palmitoylation is a key post-translational modification regulating viral replication, yet its regulatory mechanism in Dengue virus (DENV) infection remains elusive. This study aimed to elucidate the underlying regulatory function of palmitoylation and zinc finger Asp-His-His-Cys (ZDHHC) proteins in DENV replication and identify palmitoylated DENV proteins. We explored the function of palmitoylation in DENV replication using the palmitoylation inhibitor 2-bromopalmitate (2BP) and enhancer palmitic acid (PA), combined with qRT-PCR, western blot, confocal immunofluorescence microscopy, co-immunoprecipitation, and acyl-biotin exchange (ABE) assays. We found that 2BP promoted DENV replication, while PA inhibited it. Further analyses showed that 2BP and PA exerted no significant effects on DENV adsorption, internalization, or the interactions between E protein and the structural proteins prM/C that are essential for viral assembly. ABE assays verified that the DENV E protein is palmitoylated, with no such modification detected in prM and C proteins. Mass spectrometry and site-directed mutagenesis further revealed that the DENV E protein is palmitoylated at three cysteine residues: Cys74, Cys302, and Cys333. ZDHHC11, a characterized regulator of Zika virus E protein palmitoylation, directly interacted with DENV E protein and catalyzed its palmitoylation. Functionally, knockdown of ZDHHC11 enhanced DENV replication, while overexpression suppressed it. These findings deepen our understanding of flavivirus-host interaction mechanisms and provide a theoretical basis for the development of broad-spectrum anti-flavivirus therapies.