FIG. 5.
Static internal architecture of large HCV RCs. Live Huh-7.5-I/5A-GFP-6 cells were analyzed with an inverted Zeiss LSM 510 confocal laser scanning microscope with a Plan-Neofluar 63× NA 1.25 objective for a specified time period. Images of cells were recorded every 7 s. At the top, an overview image from the beginning of the experiment is shown. A rectangular region (dashed box) was bleached with a 488-nm laser pulse at time point t = −14 s for a duration of 14 s, as indicated in the time line. This area overlapped with one-half of a large structure representing a membranous web (dFWHM = 1.7 μm; arrow). Enlargements of the continuous line box area for the time point before bleaching (t = −14 s), immediately after bleaching (t = 0 s), and 8 min after bleaching (t = 480 s) are shown in the middle panels. Directly after bleaching, no fluorescence could be detected in the right half of the large structure. The structure slowly migrated to the left during a time period of 8 min, as indicated by the distance to the dashed box, which was drawn at the original coordinates. However, no fluorescence recovery was detected in the bleached half. The nonbleached half, which was the only portion of the large structure that remained visible, sustained its fluorescence intensity and size, which indicates that NS5A-GFP was not redistributed between the bleached and nonbleached halves. Three potential outcomes of this experiment are illustrated at the bottom, as discussed in Results. Note that the outcome illustrated in diagram c was observed in this experiment. Scale bars indicate reference distances of 5 and 2 μm, respectively.