Alphaviruses are mosquito-transmitted viruses that cause severe zoonotic diseases. Their envelope glycoproteins, E1 and E2, undergo cysteine acylation, a process critical for virus infection but previously undefined mechanistically. Using the Getah virus as a model, we found that E1 is acylated at Cys433 in the endoplasmic reticulum (ER), a modification beneficial for virus entry. E2 follows a unique stepwise acylation pattern: Cys385 is acylated in the ER, while Cys395, Cys415, and Cys416 undergo interdependent acylation in the Golgi. Palmitoylation of E2 Cys415/416 proved essential for budding. Acylation also facilitated cholesterol incorporation into virions independently of lipid rafts. A small interfering RNA (siRNA) screen identified distinct acyltransferases for E1 and compartment-specific enzymes for E2. Depletion of Golgi-localized Asp-His-His-Cys (DHHC)11, which modifies Cys415/416, significantly inhibited replication of multiple alphaviruses. This study establishes the spatiotemporal model of alphavirus glycoprotein acylation and identifies DHHC11 as a conserved target for pan-alphavirus therapeutics, with potential implications for reducing the public health burden of alphavirus infections.
