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.

Tagging 3A with GFP(S11) results in a replication-competent CVB3 that generates fluorescent replication organelles. Introduction of the GFP(S11) tag into the infectious clone of CVB3 yielded viable virus, i.e., CVB3-3A(S11aa2). Sequence analysis of the viral genome confirmed that the tag was retained in the 3A protein without mutations. In addition, we generated a replication-competent CVB3 with the short affinity tag StrepII of 10 residues in 3A [i.e., CVB3-3A(StrepIIaa2)]. Insertion of the StrepII tag also did not affect the known functions of 3A (data not shown), and the tag was retained in 3A for five passages (data not shown), further demonstrating that residue 2 is an amenable site for introduction of a small tag.