FIG. 6.

Tracking of fluorescent SARS-CoV VLPs in living cells. (A) Wide-field fluorescence microscopy images showing the accumulation of fluorescent VLPs at the plasma membrane of pIRES-M-E plus pcDNA-NeCFP cotransfected cells (panel b), whereas a strong perinuclear staining was observed in Vero E6 cells expressing NeCFP alone (panel a). (B) Confocal microscopy of living cells expressing M-E-NeGFP VLPs. Four categories of fluorescent signals were observed: a bright and static large perinuclear compartment (white encircling lines), smaller and dimmer actively trafficking vesicles (orange encircling lines), bright dots accumulating at the cell cortex (yellow encircling lines), and dots in the medium surrounding transfected cells (yellow dots). Videos are available in the supplemental material. (C) Treatment of transfected cells with BFA alters the trafficking of fluorescent vesicles. Vero E6 cells were transfected with pIRES-M-E plus pcDNA-NeGFP plasmids. Cells were either not treated (panel a) or treated with 6 mg of BFA/ml for either 4 h (panel d) or overnight (panel g). BFA was then added to untreated cells, and time-lapse acquisitions were performed. Panels b and c show the same cells as in panel a but after 25- and 70-min incubations with the drug. Alternatively, BFA was washed out and recovery after BFA treatment was analyzed (panels e and f and panels h and i). Panels a, b, and c, panels d and e, and panels g and h show the same cells at different time points. Videos illustrating panels b, f, h, and i are provided in the supplemental material.