Fig 6. ATP1A1 and PHB are involved in different steps of the mammarenavirus life cycle.
(A) Influence of time of addition of ouabain (OUA) or rocaglamide (Roc-A) on virus multiplication. A549 cells seeded (2.5 x 105 cells/well) in a 12-well plate and cultured overnight were infected (moi = 0.1) with rLCMV/eGFP or remained uninfected (mock). OUA (10 nM), Roc-A (100 nM), or DMSO (0.01%) was added to the culture media at the indicated time points and remained present throughout the end of the experiment. Ammonium chloride (20 mM) was added to culture medium at 4 h pi to prevent multiple rounds of virus infection. At 24 h pi, eGFP expression in infected cells was examined by flow cytometry. Data represent mean ± SD of the results of three independent experiments. (B) Effect of ouabain and Roc-A on LCMV replication. A549 cells seeded (1.25 x 105 cells/well) in 24-well plates and cultured overnight were infected (MOI = 1) with rLCMVΔGPC/eGFP, followed by addition of the indicated concentrations of ouabain or Roc-A. At 72 h pi, total cell lysates were prepared, and eGFP expression levels were measured using a fluorescent plate reader. Data represent mean ± SD of three independent experiments. (C-E) Effect of ouabain and Roc-A on Z-mediated budding. Cells (HEK 293T) seeded (3.5 x 105 cells/well) in a 12-well plate and cultured overnight were transfected with 0.5 μg of either pC-Empty or pC-LCMV-Z-Strep (LCMV-Z-Strep) (C) or pC-LASV-Z-FLAG (LASV-Z-FLAG) (D, E). At 24 h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag (C) and FLAG-tag (D). Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel C correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treated-samples was set to 100%. Data represent mean ± SD of four independent experiments. (F) Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded (4.0 x 105 cells/well) in a 12-well plate and cultured overnight were infected (MOI = 0.1) with scrLCMV/ZsG (1st infection) for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM (OUA). At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells (2nd infection) seeded (4.0 x 105 cells/well) in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate (2nd) by that in 293T cell lysate (1st). The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. (G) Effect of ouabain on the late stage of LCMV infection. A549 cells seeded (1.25 x 105 cells/well) and cultured overnight were infected (MOI = 0.1) with rLCMV/eGFP. At 48 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh medium in the presence of ouabain (OUA) at indicated concentrations. At 72 h pi, TCS was collected and virus titers were determined by IFFA. Data represent means ± SD from three independent experiments.