West Nile Virus Replication Requires Fatty Acid Synthesis but Is Independent on Phosphatidylinositol-4-Phosphate Lipids
Figure 3
Replication of WNV is independent of PI4P.
(A) Localization of PI4P at the Golgi complex in mock-infected cells. Cells transfected with a plasmid encoding a GFP-tagged FAPP1-PH protein to detect PI4P were fixed and processed for immunofluorescence (24 h p.i.) using WGA-AF594 and a mouse monoclonal antibody against GM130 (revealed with a secondary antibody coupled to AF647). (B) Localization of PI4P in WNV infected cells. Cells transfected as in (A) and later infected with WNV (MOI of 5 PFU/cell) were fixed and processed for immunofluorescence (24 h p.i.). WGA labelled with AF594 or a rabbit anti-calreticulin antibody was used in combination with a monoclonal antibody against dsRNA. Primary antibodies were detected using suitable AF594 or 647 labelled secondary antibodies. (C) Cells transfected as in (A) and later infected with CVB5 (MOI of 5 PFU/cell) were fixed and processed for immunofluorescence (8 h p.i.) as described in (B). (D) WNV RNA replication is independent of PI4KIIIβ function. Cells infected with WNV (MOI of 0.5 PFU/cell) were treated with different concentrations of PIK93 from 0 or 3 h p.i. throughout the rest of the assay. RNA from infected plates was extracted at 24 h p.i. and the number of WNV RNA copies was determined by quantitative RT-PCR. (E) Cells were infected with WNV or CVB5 and treated with PIK93 as in (D). Total virus yield (24 h p.i. for WNV and 8 h p.i. for CVB5) was determined by plaque assay. Scale bars: 10 µm.
