Fig 6. SINV cell-cell transmission is independent of NRAMP in target or producer cells.

(A) Down-regulation of target cell NRAMP. Vero cells were transfected with WT SINV or SFV RNA and incubated at 37°C for 5 h (producer cells). Target Vero cells stably expressing the PM-GFP marker were cultured for 3 days in control media or media containing 200 μg/ml ammonium iron citrate to down-regulate the SINV receptor NRAMP2, and then plated onto the infected cells at an approximate ratio of 1:1 and the co-cultures incubated for 19 h at 37°C in the continued presence of iron as indicated. The % infected target cells was 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 target cells (which was set to 1). (B) Down-regulation of producer cell NRAMP. Vero cells were pretreated as in Fig 6A to downregulate NRAMP2, transfected with WT SINV or SFV RNA, and incubated at 37°C for 5 h (producer cells). Uninfected Vero cells stably expressing PM-GFP (target cells) were then plated onto the infected cells, and the co-cultures were incubated for 19 h at 37°C in the continued presence of iron as indicated. Infection of target cells was quantitated as in Fig 6A. The graphs in A and B represent the mean and standard deviation of three independent experiments.