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.

While EM analyses have provided insight into the structure of the enterovirus ROs, fluorescence microscopy studies have focused on unraveling their origin by investigating the presence of essential host factors or marker proteins on the ROs. These studies have been performed in cells that had been fixed at various time points postinfection, usually at 1-to 2-h intervals, which gives limited insight into the dynamics of RO formation. ROs are mostly visualized by immunolabeling using antibodies directed against viral proteins that are anchored in the RO membrane, i.e., 2B, 2C, or 3A. With this approach, ROs were shown to colocalize with several proteins involved in endoplasmic reticulum (ER)-to-Golgi transport (22) (23) (24) (25) as well as with LC3, a protein involved in the autophagy pathway (11, 13, 19) . However, ROs are not mere remnants of the early secretory pathway or constituents of the autophagy pathway. Instead, enteroviruses seem highly selective in hijacking components from these pathways to create completely new organelles with a unique protein and lipid composition optimized for genome replication (reviewed in reference 5).