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.

Live-cell imaging reveals the dynamics of Golgi disassembly in live infected cells. Enterovirus infections trigger the disassembly of the Golgi apparatus, which coincides with the formation of early ROs (18, 22) . Here, we used live-cell imaging to visualize this process in living cells. To this end, BGM cells stably expressing GFP(S1-10) were transduced with murine leukemia virus (MLV) particles encoding mCherry-GM130 as a traceable marker for the Golgi apparatus. Live-cell confocal imaging was first carried out with a narrow pinhole (95.56 m) to detect the first local changes in Golgi structure during infection. As expected, GM130 was observed as a condensed perinuclear signal in uninfected cells (Fig. 5A) or early in infection with CVB3-3A(S11aa2) when fluorescent 3A (which will be further referred to as 3A-GFP) could not yet be detected ( Fig. 5B ; see Movies S1 and S2 in the supplemental material). Strikingly, the first 3A-GFP signal detected in infected cells was rarely associated with GM130, but could instead be observed as distinct cytoplasmic punctae. Localization of 3A-GFP signal to the Golgi region typically occurred within 25 min (n Ï­ 17; range, 0 to 45 min) of initial detection. This was followed by a sharp increase in the intensity and number of Golgi-adjacent 3A-GFP punctae and a local perturbation of Golgi morphology, characterized by the onset of GM130 signal fragmentation (Fig. 5B , white asterisk in the GM130 channel). Interestingly, while this rapidly increasing 3A-GFP signal was clearly detected in the Golgi region, it rarely colocalized directly with the GM130 marker (Fig. 5B, inset) , which is in agreement with previous observations in fixed cells (22) . This suggests that RO membranes originate from a Golgi compartment that is not labeled by the cis-Golgi marker GM130 or that 3A resides there only transiently before accumulating in the ROs.