West Nile Virus Replication Requires Fatty Acid Synthesis but Is Independent on Phosphatidylinositol-4-Phosphate Lipids
Figure 5
Replication of USUV is associated to the ER, requires fatty acid synthesis and is independent of PI4P.
(A) Ultrastructure of USUV-induced membrane alterations. Vero cells infected with USUV (MOI of 5 PFU/cell) were fixed and processed for electron microscopy at 24 h p.i. (a) and (b) Electron micrographs showing membrane alteration on USUV infected cells: induced-vesicles (Ve), vesicle packets (VP), and electron dense virions (Vi). Black arrows indicate the point of contact between a vesicle and the outer membrane of the VP. Asterisks denote Ve with electron dense fibrous material. (c) Whorls of stacked membranes. Scale bars: 200 nm. (B). Cells infected as in (A) were fixed and processed for immunofluorescence using a rabbit anti-calnexin antibody combined with a monoclonal antibody against dsRNA and WGA labelled with AF594. (C) Cells transfected with plasmid encoding an mRFP1 version coupled to an ER retention signal (IgLdR1kdel) were infected with USUV as is (A) and then fixed and processed for immunofluorescence using a monoclonal antibody against dsRNA. (D) Cells transfected with plasmid encoding a GFP-tagged FAPP1-PH protein (to detect PI4P) were infected with USUV as is (A) and then fixed and processed for immunofluorescence using a monoclonal antibody against dsRNA (E) USUV-infected Huh-7 cells were stained using rabbit anti-FASN antibodies combined with a monoclonal antibody against dsRNA. Suitable secondary antibodies coupled to AF-488, 594 or 647 were used in (B), (C), (D) and (E). (F) Vero cells infected with USUV (MOI of 0.5 PFU/cell) were treated with 1 µM PIK93, 15 µM cerulenin, or 15 µM C75 from 3 h p.i. throughout the rest of the assay, and total virus yield (24 h p.i.) was determined by standard titration in semisolid medium. Scale bars: 10 µm.
