Phospholipase A2 activity during the replication cycle of the flavivirus West Nile virus
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).