Fig 5. Formation of intercellular extensions and virus cell-cell transmission are independent of NRAMP2.

(A) Effect of NRAMP downregulation on free virus infection. Vero cells were cultured for 3 days in control media or media containing 200 μg/ml ammonium iron citrate to down-regulate the SINV receptor NRAMP2. Cells were infected with SINV or SFV (MOI = 5) for 2 h at 37°C. 20 mM NH₄Cl was then added to the medium to prevent secondary infection. Cells were fixed at 24 h post-infection and the ratio of infected to total cells quantitated by staining with antibody to the SINV or SFV E2 protein. The graph represents the mean and standard deviation of three independent experiments, with infection normalized to that of control cells (which was set to 1). * P<0.05. (B) Effect of NRAMP downregulation on formation of intercellular extensions. Vero cells were infected with SINV (MOI = 5) for 5 h at 37°C. Target Vero cells stably expressing the PM-GFP marker were pretreated as in (A) to downregulate NRAMP2, and then plated onto the infected cells at an approximate ratio of 1:1. The co-cultures were incubated for 3 h at 37°C in the continued presence of iron as indicated, fixed, permeabilized, and stained with antibody to SINV E2. Fluorescence microscopy images were acquired with the same exposure time, and images are representative of the results of 3 independent experiments. Bar = 20 μM. (C) Quantitation of the number of extensions in Vero cells from experiments as described in (B). Intercellular extensions emanating from infected cells were identified based on positive staining for the SINV E2 protein and contact with a PM-GFP expressing target cell, as described in the methods. The graph in (C) represents the mean and standard deviation of three independent experiments.