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.

Enterovirus ROs were imaged in living BGM(GFPS1-10) cells upon infection with CVB3-3A(S11aa2). As a proof of concept, we also expressed GM130-mCherry in these cells to visualize Golgi disassembly in real time. The onset of Golgi fragmentation was found to correspond to a period of rapid 3A accumulation in the Golgi region. Under the experimental conditions used, Golgi disruption was completed within 75 to 105 min after the first detection of 3A-GFP. Interestingly, while 3A accumulation occurred adjacent to the GM130 signal, 3A rarely colocalized with GM130-mCherry before and during Golgi disassembly. While this does not preclude the transient localization of 3A to the cis-Golgi, our findings suggest that ROs may be generated from another Golgi compartment not labeled by GM130, which is in line with previous observations that suggest that RO formation is initiated at the trans-Golgi network (22) .