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.

Live-cell imaging is a powerful technology to gain insight into the dynamics of biological processes. In the field of virology, this method has mostly been applied to visualize the entry and egress pathways of viruses by following the fate of fluorescently labeled, individual virus particles in living cells and monitoring their interactions with cellular structures (52) . To date, imaging replication structures during infection has been reported for only a few viruses, including hepatitis C virus (53, 54) , vaccinia virus (55), turnip mosaic virus (56), mouse hepatitis virus (57, 58) , and equine arteritis virus (59) . In most of these studies, replication structures are illuminated by a viral protein that is fused to GFP. Recombinant enteroviruses that encode an RO-anchored viral protein fused to GFP (or another fluorescent reporter) have not been reported thus far. On the other hand, small epitope tags have been successfully introduced into the 3A protein of poliovirus (32) , which prompted us to test whether small tags suitable for fluorescent labeling are accepted in the 3A protein of CVB3. The smallest tag for fluorescent labeling of a protein is the tetracysteine tag of 6 to 20 residues (depending on the version), but it requires visualization with biarsenical dyes FlAsH and ReAsH in extra labeling steps and results in fluorescence with a relatively poor quantum yield (60, 61) . The split-GFP system uses a tag of 16 residues, GFP(S11), which has the advantage that it does not require staining but becomes directly fluorescent upon assembly with the large GFP(S1-10) fragment in living cells (34) . This system has been successfully applied before in the context of a virus to study the intracellular trafficking of ribonucleoproteins of influenza A virus, where the PB2 polymerase subunit was tagged with GFP(S11) (62) . In our study, we incorporated GFP(S11) into the RO-anchored 3A protein of CVB3. We show that the introduction of GFP(S11) after the second residue does not affect the localization or function of 3A when it is expressed in isolation. Whether binding of GFP(S1-10) affects the function of 3A(S11aa2) proteins remains unknown, as it is possible that not all 3A(S11aa2) proteins are bound by the GFP(S1-10) counterparts. Therefore, the subset of unbound 3A(S11aa2) proteins could be solely responsible for exerting the 3A functions.