Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na+/K+ transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses
Fig 5
Effect of pharmacological inhibition of ATP1A1 and PHB on LCMV multiplication.
(A) A549 cells seeded (1.25 x 105 cells/well) in 24-well plates and cultured overnight were treated with either ouabain (OUA) (i) or rocaglamide (Roc-A) (ii) at indicated concentrations or with DMSO (vehicle control) for 2 h and then infected (MOI = 0.01) with rLCMV/eGFP. Compounds were present throughout the experiment. At 24 and 48 h pi, TCSs were collected, and virus titers determined by IFFA. Data represent means ± SD of results from three independent experiments. LoD, the limit of detection. (B) Inhibitory effects of ouabain and Roc-A on virus propagation and cell viability. A549 cells seeded (2.0 x 104 cells/well) in 96-well plates and cultured overnight were treated with 3-fold serial dilutions of either ouabain (i) or Roc-A (ii) for 2 h and then infected (MOI = 0.01) with rLCMV/eGFP. Compounds were present throughout the experiment. At 48 h pi, cells were fixed to examine eGFP expression and cell viability as determined by CellTiter 96 AQueous one solution reagent. The data represent means ± SD of the results from four (cell viability assay) or six (virus spread assay) replicates. The therapeutic index (TI) was calculated by dividing CC50 by IC50. (C) Effect of ouabain or Roc-A treatment on rVSV/eGFP multiplication. A549 cells seeded (1.25 x 105 cells/well) and cultured overnight were treated with either ouabain (10 nM) Roc-A (100 nM), or vehicle control (DMSO) for 2 h and infected (MOI = 0.01) with either rLCMV/eGFP or rVSV/eGFP. At 72 h pi, TCS was collected, and virus titers were determined by IFFA (rLCMV/eGFP, expressed as FFUs) or a plaque assay (rVSV/eGFP, expressed as PFUs). Compounds were present to study endpoint. Results represent means ± SD of the results of three independent experiments.