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

Figures

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  Fig. 1. RNAseK is S-palmitoylated at four cysteine (Cys) residues. (A) Amino acid sequence of RNAseK. Transmembrane domains are underlined. RNAseK contains five cysteine residues. Mass spectrometry analysis identified S-palmitoylation at Cys6, Cys7, and Cys85 (highlighted in yellow, see also Figure S1) [20]. Cys14 and Cys69 are highlighted in green and purple, respectively. (B) Schematic representation of the IP/Click assay. Cells were metabolically labelled with alk-16, an alkyne-modified palmitate analogue, followed by immunoprecipitation (IP) and copper-catalysed azide-alkyne cycloaddition (CuAAC) to couple azido-TAMRA-biotin for detection. (C) Cells transfected with empty vector (EV), RNAseK-FLAG-WT or Cysteine-to-Alanine mutants were subjected to the IP/Click assay. Input: whole cell lysate. Equal amounts of total protein were loaded per condition (GAPDH, loading control). Data shown are representative of biological duplicates. (D) Schematic representation of the Acyl-PEG exchange (APE) assay [30]. Free Cys are first blocked with N-ethylmaleimide (NEM). S-palmitoyl groups are then cleaved with hydroxylamine (NH₂OH), revealing previously modified cysteines, which are subsequently…

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  Fig. 2. S-palmitoylation of RNAseK is relevant for ZIKV infection. A549 cells stably expressing either RNAseK-FLAG-WT or RNAseK-FLAG-∆Palm, A549 control cells, A549 shRNAseK knockdown cells and A549 cells treated with Bafilomycin A1 (10 nM) or Dynasore (100 µM), were infected with ZIKV at a multiplicity of infection (MOI) of 1 for 72 h. Subsequently, culture supernatants were collected for RT-qPCR and TCID50 assays, while cells were harvested for flow cytometry analysis. (A) The barplot depicts the percentage of Zika virus-infected cells detected by flow cytometry analysis at 72 hpi. Infected cells were stained with an anti-Flavivirus Envelope antibody. Data are presented as mean ± standard deviation from three independent biological replicates. ND = Non-Detectable. A one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons (p < 0.01) was performed. Asterisks indicate significant differences between experimental conditions and the control A549 cells. (B) Barplot representing the viral RNA levels determined by RT-qPCR for each condition. Data are presented as mean ± standard deviation from three independent biological replicates. ND = Non-Detectable. Data were normalised…

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  Fig. 3. Subcellular localisation of RNAseK-FLAG variants. (A) HEK293T cells were transiently transfected with plasmids encoding either RNAseK-FLAG-WT or RNAseK-FLAG-ΔPalm. Cells were fixed 24 h post-transfection and subjected to immunofluorescence assays to detect RNAseK variants along with organelle-specific markers. Representative images are shown: RNAseK-FLAG (green), organelle markers (red), and nuclei stained with DAPI (blue). Colocalisation between RNAseK-FLAG and organelle markers is indicated in yellow. Scale bar, 10 µm. (B) Quantitative colocalisation analysis between RNAseK-FLAG variants and the different organelle markers. Box plots show Mander’s coefficients for each RNAseK-FLAG/marker pair. At least 30 fluorescence-positive images per coverslip were analysed for each condition. A one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons (p < 0.01) was performed. No significant differences were observed between RNAseK-FLAG-WT and RNAseK-FLAG-ΔPalm under any evaluated condition.

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  Fig. 4. S-palmitoylation of RNAseK prevents its degradation by the proteasome (A) HEK293T cells were transfected with plasmids encoding either RNAseK-FLAG-WT, RNAseK-FLAG-ΔPalm or the empty vector (-). 24 h post-transfection, cells were treated with Cycloheximide (10 µg/mL) for 0, 6, 12, or 24 h. Subsequently, cells were harvested and whole-protein lysates were separated by SDS-PAGE. Overexpressed RNAseK-FLAG-WT and ΔPalm were detected by Western blot using an anti-FLAG antibody. Equal amounts of total protein were loaded per condition. Alpha-tubulin was used as a loading control. Results are representative of three independent biological replicates. (B) A549 cells stably expressing either RNAseK-FLAG-WT or RNAseK-FLAG-∆Palm as well as A549 control cells and A549 shRNAseK knockdown cells, were treated with either vehicle control (DMSO) or the proteasome inhibitor MG-132 (10 µM) for 24 h. Subsequently, cells were harvested and whole-protein lysates were separated by SDS-PAGE. Proteins of interest were detected by Western blot using specific antibodies. Equal amounts of total protein were loaded per condition. Alpha-tubulin was used as a loading control. Results are representative of three…

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  Fig. 5. Effect of RNAseK S-palmitoylation on lysosomal acidification assessed by live-cell imaging. (A) Schematic illustration of the ratiometric lysosomal pH reporter pHLuorin-LAMP1-mApple. (B) Representative live-cell fluorescence microscopy images of A549-derived cell lines stably expressing the indicated RNAseK variants and transiently transfected with pHLuorin-LAMP1-mApple. mApple fluorescence is shown in red, pHLuorin fluorescence in green, and merged signals in yellow. Scale bar, 10 µm. (C) Quantitative analysis of fluorescence signals from mApple (MI₅₉₂) and pHLuorin (MI₅₁₁). The ratio of pHLuorin (MI₅₁₁) to mApple (MI₅₉₂) fluorescence intensity was calculated for each condition and normalized to parental A549 cells. Bar graphs show mean ± SD from three independent biological replicates. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons (p < 0.01). Asterisks indicate statistically significant differences relative to parental A549 control cells. Increased pHLuorin/mApple ratios indicate reduced lysosomal acidification.

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  Fig. 6. S-palmitoylation of RNAseK increases upon ZIKV infection, while total RNAseK levels decrease. Time-course of RNAseK S-palmitoylation after ZIKV infection. An acyl-biotin exchange assay was performed to assess S-palmitoylation levels of RNAseK-FLAG-WT after 0- 72 hpi of ZIKV infection in A549 cells stably expressing RNAseK-FLAG-WT. NI refers to non-infectedcells processed in parallel, whereas 0 hpi corresponds to infected cells harvested immediately after the 2 h infection period (virus adsorption and entry) and medium replacement, prior to significant viral replication. (A) Western blots showing S-palmitoylated (S-palm) RNAseK-FLAG-WT and overall RNAseK-FLAG-WT protein (Input). (B) S-palmitoylated RNAseK-FLAG-WT values in the graph were derived from S-acylated RNAseK-FLAG-WT normalised to RNAseK-FLAG-WT “Input” and β-actin as a loading control. (NI = non-infected). Data are presented as mean ± standard deviation from four independent biological replicates, with individual data points shown. Statistical comparisons were performed using an unpaired Student’s t-test versus the NI condition: * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.