(Row A) HeLa cells were infected with VACV and after 2 h processed and stained with anti-E3 mouse MAb (red), followed by Alexa Fluor 488 goat anti-mouse IgG and DAPI (blue). Two infected and several uninfected cells are present in the field. (Row B) Same as Row A except that cells were processed at 6 h after infection. (Row C) VACV infected HeLa cells were fixed after 8 h, processed and stained with rabbit polyclonal anti-A23 (red), followed by Alexa Fluor 488 goat anti rabbit IgG and DAPI (blue). F, virus factory.

(Row A) Uninfected (Un) HeLa cells were fixed and stained with goat polyclonal anti-eIF4G (green), Alexa Fluor 488 donkey anti-goat IgG and DAPI (blue). VACV infected (In) HeLa cells were processed after 8 h and stained sequentially with goat polyclonal anti-eIF4G (green), mouse MAb to G3BP (red), Alexa Fluor 488 donkey anti-goat IgG, Alexa Fluor 594 goat anti-mouse IgG and DAPI (blue) (Row B) Uninfected (Un) HeLa cells were stained with mouse MAb to eIF4E (green), Alexa Fluor 488 goat anti-mouse IgG and DAPI (blue). VACV infected (In) HeLa cells were stained sequentially with mouse MAb to eIF4E (green), rabbit polyclonal anti-G3BP (red), Alexa Fluor 488 goat anti-mouse IgG, and Alexa Fluor 594 goat anti-rabbit IgG.

(Row A) HeLa cells were infected with VACV for 6 h. The cells were then washed, fixed, stained with acridine orange (AO) and viewed by confocal microscopy to visualize DNA (green) and RNA (red). One entire cell (center) and the cytoplasm of a second cell (bottom) are seen. The green stain outside the cell is due to AO bound to the glass cover slip. N, nucleus; C, cytoplasm; F, viral factory. (Row B) Uninfected HeLa cells were transfected with anti-sense G8R RNA labeled with digoxigenin UTP. After 6 h, the cells were washed, fixed and stained with sheep anti-digoxigenin-fluorescein, followed by Alexa Fluor 488 donkey anti-sheep (green) and DAPI (blue). (Row C) HeLa cells were infected with VACV and transfected 1 h later with anti-sense G8R RNA labeled with digoxigenin UTP. At 6 h after infection, the cells were analyzed as in Row B. (Row D) Uninfected HeLa cells were transfected with biotinylated poly (U). After 6 h, the cells were stained with streptavidin Alexa Fluor 488, followed by anti-Alexa Fluor 488 rabbit IgG (green) and DAPI (blue). (Row E) HeLa cells were infected with VACV and transfected 1 h later with biotinylated poly (U) and processed as in Row D.

(Row A) Uninfected HeLa cells were fixed and sequentially stained with mouse MAb anti-G3BP (green), rabbit polyclonal anti-p137 (red), Alexa Fluor 488 goat anti-mouse IgG, and Alexa Fluor 594 goat anti-rabbit IgG and DAPI (blue) and viewed by confocal microscopy. (Row B) HeLa cells were infected with VACV and after 8 h were processed as in Row A. (Row C) HeLa cells were infected with VACV and transfected 1 h later with biotinylated poly (U). After 6 h the cells were fixed and stained sequentially with streptavidin Alexa Fluor 488 (green), mouse MAb anti-G3BP (red), anti-Alexa Fluor 488 rabbit IgG fraction, Alexa Fluor 594 goat anti-mouse IgG and DAPI (blue).

(Rows A, B) HeLa cells were infected with 1 PFU per cell of VACV expressing β–Galactosidase driven by the G8R intermediate promoter. After 6 h the cells were fixed and processed for confocal microscopy. Cells were stained with rabbit polyclonal anti β–Galactosidase (green), followed by Alexa Fluor 488 goat anti-rabbit IgG and DAPI. In row B, the nuclei and adjacent factories are in three separate cells (Rows C–F) Hela cells were infected with 0.5 PFU of vA5L-Cyan and 0.5 PFU of vA5L-Yellow. After 8 to 12 h, the cells were fixed and florescence was monitored at the appropriate channels using confocal microscopy. In row D, the field contains one infected cell with factories and parts of several uninfected cells.