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