Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

van der Schaar, H. M.; Melia, C. E.; van Bruggen, J. A. C.; Strating, J. R. P. M.; van Geenen, M. E. D.; Koster, A. J.; Bárcena, M.; van Kuppeveld, F. J. M.

stained with MitoTracker deep red FM, and monitored by live-cell imaging. Full-depth z-stacks of cells emitting GFP fluorescence were taken, and cells were processed for EM. Light microscopy (LM)-EM overlays were made using MitoTracker deep red FM as an orientation guide, and 3A-GFP signal was aligned to the corresponding EM image in this manner first at low magnification (A) and then within individual cells of interest (B) (panel B is the boxed area in panel A). 3A-GFP signal was found at the typical structures that develop during CVB3 infection, including single-membrane tubules (red box in panel B, enlargement shown on the right) and double-membrane vesicles (black box in panel B, enlargement shown on the right). Bars, 30 m (A) and 5 m (B).