Fig. 4

The rac-1 and rho A kinases are required for localised cell-to-cell transmission in the HEp2 cell monolayer. a Mock and RSV-infected cells (multiplicity of infection (moi) of 0.1) were stained using anti-F and anti-rac-1 at 24 h post-infection (hpi) and imaged by confocal microscopy (objective x100 magnification). Only the merged image is shown in the mock-infected cells. b Co-stained cells showing virus filaments bridging two cells. In all cases the virus filaments are highlighted (white arrows). c Virus particles were dissociated from infected cells and infectious virus particles isolated using discontinuous sucrose gradient centrifugation as described previously [21]. c (i) A representative gradient showing the locations of the fractions at the 20-35 % sucrose (band-1), the 35-45 % sucrose (band-2) and 45-55%sucrose (band-3) interfaces. (ii) Each fraction was examined by immunoblotting using anti-rac-1. A protein band corresponding in size to the rac-1 protein is highlighted (black arrow). d and eThe rac-1 inhibitor NSC23766 prevents virus filament formation and virus transmission. d RSV-infected HEp2 cells (moi = 0.1) were either non-treated or NSC23766-treated and the cells stained using anti-G. The virus filaments in non-treated (white arrows) and punctuate staining pattern in NSC23766-treated cells (*) are highlighted. In each case (ii) is an enlarged image of the region highlighted by the white box in (i). e rac-1 activity is required for cell-to-cell transmission. HEp2 cell monolayer were infected using a moi of 0.0002 and at 5 hpi the cell were either non-treated or NSC23766 treated. At 18 hpi the inhibitor was either removed (washout) or maintained. At 36 hpi the cells were fixed and stained using anti-RSV. The cells were imaged using immunofluorescence microscopy (objective x20 magnification). The infected cell clusters in the NT cells (open white box) and smaller clusters in the NSC23766-treated cells (white arrows) are indicated. f HEp2 cell monolayers were infected with RSV using a moi of 0.0002 and at 18 hpi the cells were either non-treated (NT) or treated with Y-27632 (YT). At 48 hpi the cells were fixed and stained using anti-RSV and imaged using immunofluorescence microscopy (objective x20 magnification). The infected cell clusters in the NT cells (open white box) and smaller clusters in the YT cells (white arrows) are indicated