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.

Having produced replication-competent CVB3-3A(S11aa2), we then tested whether this virus induced GFP fluorescence in cells expressing GFP(S1-10). Figure 3 shows that these cells do emit GFP fluorescence upon infection with CVB3-3A(S11aa2). To investigate whether the GFP fluorescence was restricted to the sites where the 3A(S11aa2) proteins are localized in the cell, we first stained cells with an anti-GFP antibody. Figure 3A shows that the anti-GFP antibody recognized GFP(S1-10) as demonstrated by the cytoplasmic staining in uninfected cells, whereas BGM cells lacking GFP(S1-10) were negative (data not shown). In infected cells, GFP fluorescence was not observed throughout the cytoplasm but in a pattern that is reminiscent of a 3A staining pattern. This suggests that assembled, fluorescent GFP is found only at sites that contain BGM cells stably expressing GFP(S1-10) were infected with CVB3-3A(S11aa2). At 5 and 6 h p.i., cells were fixed and subjected to immunofluorescence analysis. The detected GFP fluorescence is the result of GFP(S1-10) assembling with 3A(S11aa2). Nuclei were visualized with DAPI. Images were acquired with a Leica SPE-II DMI-4000 confocal laser scanning microscope. (A) GFP(S1-10) was detected with a primary polyclonal antibody directed against GFP and a secondary Alexa Fluor 594-labeled antibody. The uninfected cell is marked with a white asterisk. (B) The 3A(S11aa2) protein was stained with a primary antibody directed against 3A and a secondary Alexa 3A(S11aa2). Interestingly, while anti-GFP antibody staining in infected cells was detected throughout the cytoplasm, the signal was more intense at regions corresponding to GFP fluorescence. This is likely the result of recruitment of GFP(S1-10) to sites where 3A is localized. Another possibility is that the anti-GFP antibody has a higher affinity for full-length, assembled GFP.