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.

In this study, we used a split-GFP system (34) to tag the 3A protein of CVB3, a small protein of 89 amino acids with a C-terminal hydrophobic domain that inserts into the RO membranes (35) . This split-GFP system is based on superfolder GFP, a variant of GFP that folds better, matures more rapidly, and fluoresces more brightly than GFP (36) . Like GFP, superfolder GFP has a beta barrel structure consisting of eleven ␤-strands, which in this system are split into a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 amino acids. Separately, these two fragments are nonfluorescent. Green fluorescence is emitted only when they assemble into superfolder GFP (34) . We show that tagging 3A with GFP(S11) does not affect its known functions when 3A is expressed alone. When introduced into the viral genome of CVB3, GFP(S11)-tagged 3A assembled with GFP(S1-10) to illuminate enterovirus ROs as shown by correlative light electron microscopy (CLEM). We illustrate the suitability of CVB3 encoding this split-GFP-tagged 3A for live-cell imaging by monitoring the development of ROs and the disintegration of the Golgi apparatus, as visualized using GM130-mCherry, in infected cells in real time. This new tool simplifies the visualization of enterovirus ROs, avoiding immunolabeling of viral proteins in fixed cells, and will have multiple applications in future studies on the origin, location, and function of enterovirus ROs.