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.

3A tagged with GFP(S11) assembles with GFP(S1-10) to yield GFP fluorescence. In order to visualize the enterovirus ROs in living cells, one of the nonstructural proteins that is anchored in the membranes of the ROs should be labeled with a fluorescent tag. A transposon-based insertion mutagenesis study revealed that the N-terminal region of poliovirus 3A tolerates small insertions (32, 37) . Specifically, viable insertions were found after residues 2, 6, 9, 10, and 11. Since the 3A proteins of coxsackievirus B3 (CVB3) and poliovirus are highly homologous, these positions were good candidates for introduction of a small tag in CVB3 3A. We chose to insert GFP(S11) after amino acid 2 in CVB3 3A without using any linker residues, yielding 3A(S11aa2) (Fig. 1A) .