Fig. 3.

Brefeldin A reduces surface nucleoprotein expression and increases N protein accumulation within the Golgi compartment. (A) Flow cytometry analyses of BFA- and WM-inhibition assays. At 24 hpi, control and hRSV-infected (MOI = 5) HEp-2 cells were incubated for 5 h with either BFA (10 μg/mL) or WM (500 nM), washed twice, and analyzed by flow cytometry for expression of surface hRSV N. One group for each drug (along its vehicle control) was washed and left in culture to determine N expression in the cell surface at 48 hpi (n = 3). (B) HEp-2 cells were coinfected with hRSV (MOI = 5) and a baculovirus encoding a fusion RFP-GALNT2 (particles per cell = 30). At 24 hpi, HEp-2 cells were incubated for 5 h with BFA (10 μg/mL), DMSO (vehicle), or media (untreated) and then fixed with 4% paraformaldehyde (PFA) in PBS, permeabilized with 0.2% Triton X-100 in PBS, and stained using a directly conjugated anti-N 1E9/D1 Alexa Fluor 647 (depicted in green) (n = 3). Open arrowheads (white) indicate N-hRSV–RFP colocalization in untreated and vehicle-treated cells. Thin arrows indicate accumulation of N within the RFP-GALNT2⁺ compartment in BFA-treated cells. *P < 0.05; ***P < 0.001. n.s., nonsignificant.