S-palmitoylation of the v-ATPase subunit RNAseK is necessary for Zika virus infection

BACKGROUND: S-palmitoylation is a reversible and dynamic lipid post-translational modification that regulates protein localisation, stability, and function. During viral infections, S-palmitoylation can modulate viral replication by directly modifying viral proteins or by altering host proteins involved in the viral life cycle and immune responses. In this study, we investigated the role of S-palmitoylation of Ribonuclease K (RNAseK) during infection with Zika virus (ZIKV), a member of the flavivirus family. RNAseK is an 11 kDa transmembrane protein recently identified as a component of the vacuolar ATPase complex and required for efficient infection by flaviviruses. METHODS: We used site-directed mutagenesis, metabolic labelling with alkyne-tagged palmitate, and the Acyl-PEG Exchange (APE) assay to identify S-palmitoylation sites on RNAseK. A549 cells stably expressing wild-type or S-palmitoylation-deficient RNAseK mutants were infected with ZIKV. Viral replication was assessed by flow cytometry, RT-qPCR, and TCID₅₀ assays. Immunofluorescence was used to evaluate the role of S-palmitoylation in protein localisation. Cycloheximide chase and proteasome inhibition assays were used to assess protein stability, while lysosomal acidification assays were used to assess lysosomal pH changes. RESULTS: RNAseK was found to be S-palmitoylated on four cysteine residues (Cys6, Cys7, Cys14, and Cys85). Substitution of these residues with alanine abolished protein S-palmitoylation. Cells stably expressing the S-palmitoylation-deficient RNAseK mutant exhibited decreased ZIKV replication and reduced production of infectious viral particles compared to wild-type RNAseK-expressing cells. S-palmitoylation was required for RNAseK stability and reduced its proteasomal degradation, but did not affect its cellular localisation. S-palmitoylation of RNAseK also contributed to lysosomal acidification, a process important for ZIKV infection. CONCLUSIONS: This study identifies RNAseK S-palmitoylation as a determinant of protein stability and efficient ZIKV infection in human cells. More broadly, it highlights the importance of lipid post-translational modifications in host-pathogen interactions and suggests that targeting RNAseK S-palmitoylation may represent a potential antiviral strategy against ZIKV.