Visualization of Intracellular Movement of Vaccinia Virus Virions Containing a Green Fluorescent Protein-B5R Membrane Protein Chimera

Synthesis of B5R-GFP. HeLa cells were infected with WR or vB5R-GFP. At various time points, cells were harvested and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with MAb 19C2 to B5R. The numbers above the lanes indicate the hour after infection that the cells were harvested. The masses (in kilodaltons) and positions of marker proteins are shown on the left.

Confocal microscopy of cells infected with vB5R-GFP. HeLa cells were infected with WR (A) or vB5R-GFP (B to F) and then stained with MAb 19C2 against B5R followed by Cy5-conjugated donkey anti-rat antibody (appearing white [A] or red [E and F]). F-actin was visualized with rhodamine-phalloidin (red [A, C, and D]). GFP fluorescence appears green (B, D, and F). (D) Overlay of panels B and C; (F) overlay of panels B and E. Overlapping red and green signal is represented by yellow (D and F). Arrowheads indicate virions on the end of actin tails. Arrowheads in panels B and D to F point to the same set of virions.

Immunogold electron microscopy of cells infected with vB5R-GFP. RK₁₃ cells that had been infected with vB5R-GFP for 24 h were fixed in paraformaldehyde, cryosectioned, and incubated with GFP polyclonal antibody followed by 10-nm-diameter gold particles conjugated to protein A. IMV (A), IEV (B), and EEV (C) are shown. Scale bars, 500 nm.

Visualization of IEV by time-lapse confocal microscopy. At 1 h after infection of HeLa cells with 0.2 PFU of vB5R-GFP per cell, medium containing rifampin (0.1 μg/ml) was added to the cell monolayer. Approximately 12 h later, the rifampin was removed by washing with fresh medium. (A) Cells were viewed by confocal microscopy 2 h and 40 min later, and an image of one cell was collected every 10 s for 2 min. (B) After an additional 2 h, an image of the same cell was collected every 10 s. (C and D) HeLa cells were infected with 0.2 PFU of vB5R-GFP in the absence of rifampin, and imaging at 1 frame/6 s was started approximately 12 h later. In the upper left corner, the cumulative time elapsed (in seconds) after the start of image collection/video frame number is indicated. Arrowheads in each row point to the same IEV particle. In row B, the arrows point to virions extended from the cell on microvilli. In row D, the arrows point to tubules. N, nucleus. Scale bars, 50 μm. (The entire time-lapse videos are available athttp://www.niaid.nih.gov/dir/labs/lvd/moss.htm.)

Effect of rifampin on virion movement. HeLa cells were incubated continuously in medium containing 0.1 μg of rifampin/ml starting 1 h prior to infection with vB5R-GFP. Cells were imaged by confocal microscopy at ∼13 h after infection. Shown is a representative cell starting at time zero. Subsequent frames are shown with the cumulative time elapsed (in seconds) indicated in the upper left corner.

Visualization of IEV acquiring an actin tail. HeLa cells were infected and examined by confocal microscopy as described in the legend to Fig. 5. One image was acquired every 6 s. As in Fig. 5, the cumulative time elapsed/video frame number of the sequence is indicated in the upper left corner of each image, starting at time/frame 0/1. The frames in the first and third columns depict GFP fluorescence; the frames in the second and fourth columns depict images acquired simultaneously by the transmitted-light detector. Arrowheads point to the same virion in all frames. Arrows point to additional actin tails. Scale bar, 50 μm. (The entire time-lapse videos are available athttp://www.niaid.nih.gov/dir/labs/lvd/moss.htm.)