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.

Next, we set out to confirm that the emerging GFP fluorescence is emitted from ROs that contain 3A(S11aa2). To this end, we applied correlative light electron microscopy (CLEM) (49, 50) , which provides a direct link between fluorescent signals and the virus-induced structures that underlie them at high resolution. The emerging fluorescent signal in BGM(GFPS1-10) cells infected with CVB3-3A(S11aa2) and stained with MitoTracker deep red FM was monitored by live-cell imaging. Cells were imaged and fixed at an intermediate time point in infection. Following sample processing and EM imaging, the MitoTracker signal and corresponding mitochondria were used to orient fluorescence and EM images with an independent marker. Using this method, 3A(S11aa2) was found to localize to ROs, evident as both tubules and clusters of DMVs and multilamellar structures (Fig. 4) . Furthermore, the CLEM results not only show that GFP fluorescence was emitted from bona fide ROs, they also demonstrate that the GFP(S11) insertion in 3A does not affect the development of the different RO morphologies typically observed during enterovirus infections.