Fig 1.
LC/MS analysis of WNVKUN-infected cells reveals a homeostatic change in phospholipid levels.
Vero cells were mock- or WNVKUN-infected and harvested at 24 h.p.i. Levels of phospholipids were quantitated by LC/MS and the fold change plotted on GraphPad Prism 6. The analysis was performed on triplicate samples from triplicate experiments. (A) Lyso-phosphatidylcholine (Lyso-PChol), (B) Phosphatidylcholine (PChol), (C) Phosphatidylethanolamine (PE), (D) Phosphatidylserine (PS), (E) Phosphatidylinositol (PI). (F) Summary of significant changes in individual phospholipid species, and (G) Summary of fold change per phospholipid class.
Fig 2.
Host enzyme PLA2 are activated in WNVKUN-infected cells, and exogenous addition of lyso-PChol lipid can rescue chemical PLA2 inhibition.
(A) Summary of the homeostatic change in phospholipids as quantified by LC/MS revealing a correlation of present PChol and lyso-PChol lipid species. (B) Cellular PLA2 activity level in mock- and WNVKUN-infected cells from various cell lineages showing increased PLA2 activity during viral infection for all cell types (n = 3 independent experiments). (C) Visualisation of exogenous administered fluorophore-tagged lyso-PChol lipid moieties (lyso-PChol488; 5μM), co-stained with antibodies recognising viral NS3 protein. Bar = 2μm (images i-ii, vi-vii) and bar = 1μm for inset images (iii-v, xiii-x). Arrows indicate colocalisation. (D) Visualisation of lyso-PChol488 (5μM) co-stained with antibodies recognising replication intermediates (dsRNA). Bar = 5μm (images xii-xii, xvi-xvii) and bar = 2μm for inset images (xiii-xv, xviii-xx). Arrows indicate colocalisation. For rescue experiments, vero cells were infected with WNVKUN for an hour and ACA (20μM) drug-treated until 8 h.p.i. Exogenous lyso-PChol (1.5μM) was subsequently added and cells subject to analysis at 24 h.p.i. (E) Restored production of secreted infectious virus particles following the addition of lyso-PChol as determined by plaque assay (n = 3 independent experiments). (F and G) Western blot analysis and quantification of viral protein levels during PLA2 enzyme inhibition and lyso-PChol addition. Envelope (Env) and NS5 proteins were normalized to the cellular protein Calnexin (n = 3 independent experiments).
Fig 3.
Evaluation of the effect of fatty acid synthase and PLA2 activity inhibitors on WNVKUN replication.
Vero cells were pre-treated with the chemical inhibitors and subsequently infected for an additional 24 hrs. (A and B) Production of viral protein was determined by western blotting with mouse monoclonal antibodies against the WNVKUN envelope (Env) and NS5 proteins and normalized to cellular proteins GRP78 and actin. Quantitation is depicted in panel B. (C) Production of genomic viral RNA was determined by qRT-PCR after treatment and infection with inhibitors and virus at 24 h.p.i. (D) Production of infectious secreted virus was determined by plaque assay after treatment and infection with inhibitors and virus at 24 h.p.i. (E) Immunofluorescence analysis of WNVKUN-infected Vero cells at 24 h.p.i. following drug treatment. Cells were stained with mouse monoclonal anti-NS1 antibodies and viewed on a Zeiss confocal microscope. In all cases statistical analysis was performed on duplicate analysis of triplicate experiments via Students t-test on GraphPad Prism 6. C75 (final concentration of 30μM), ACA = n-(p-amylcinnamoyl) anthranilic acid (final concentration of 20μM) and PACOCF3 = palmityl trifluromethyl ketone (final concentration of 10μM). ACA and PACOCF3 were used at a final concentration of 15μM each when treated together.
Fig 4.
PLA2 enzyme inhibition induces an altered WNVKUN replication complex morphology.
Vero cells were pretreated with the vehicle solvent DMSO (A and B), or inhibitors C75 (30μM) (C and D), ACA (20μM) (E and F), PACOCF3 (15μM) (G and H) or ACA+PACOCF3 (15μM each) (I and J) and subsequently infected with WNVKUN for an additional 24 hrs before analysis by transmission electron microscopy on a Technai F30. Arrows indicate elongated vesicles and all magnification bars represent 200nm. (K) Vesicle length was measured on images gathered in Adobe Photoshop CS6 and quantitation performed in GraphPad Prism 6, statistical significance was determined by Student’s t-test on the number of vesicles visualized in duplicate experiments.
Fig 5.
Expression and activation of specific PLA2 isoforms during viral replication.
(A) Schematic representation of the 20 known PLA2 genes/isoforms, and their subcellular localisation, that are involved in the hydrolysis of PChol to lyso-PChol. (based on databases: KEGG, GeneCards, Reactome, Human Protein Atlas). (B and C) Western blot analysis and quantification of the phosphorylation status of PLA2G4A (p-PLA2G4A) during N-Ethylmaleimide (NME; 100μM for 30mins) control-treated and WNVKUN-infected cells (n = 3 independent experiments). (D and E) Confocal microscopy and quantification of enzyme translocation and phosphorylation of PLA2G4A and in NME control-treated and WNVKUN-infected cells. Images i-iii are based on antibodies recognizing total PLA2G4A protein, whereas images iv-ix are based on antibodies recognizing phosphorylated (activated) PLA2G4A (n = 4 independent experiments). Bar = 20um. Arrowheads indicate enzyme translocation towards the plasma membrane. (F) Confocal microscopy showing the partial sequestering of PLA2G4A protein populations to sites of viral protein expression (NS3 protein). Bar = 20μm for images x-xi and bar = 5μm for images xii-xiv. (G to K) The effect of targeted siRNA-mediated gene silencing of PLA2G4A or PLA2G4C-encoded enzymes on WNV replication. (G) Representative Western Blots and (H) quantification of viral protein levels. (I) Virus production and (J) viral RNA genomes. (K) PLA2G4A and PLA2G4C protein expression levels (n = 3 independent experiments).