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.

Although we are learning more and more about the morphology and origin of enterovirus ROs by three-dimensional EM studies and by fluorescence microscopy of fixed cells, it has not been possible thus far to directly visualize ROs in living cells. Live-cell imaging can provide unprecedented insights into the dynamics of biological processes, including RO formation and viral effects on other cellular structures or organelles, and may capture rare or rapid events that may be missed in the analysis of fixed cells. Moreover, observations by immunolabeling of fixed cells may be compromised by artifacts induced during sample preparation and should be complemented by live-cell imaging (26) . To date, enterovirus ROs have been monitored only indirectly in living cells that expressed a fluorescently labeled cellular protein, the Golgi-resident Arf1-RFP (red fluorescent protein [RFP]-labeled Arf1), as an RO marker (22) . Yet, the use of a cellular protein as a RO marker may complicate investigations examining the transition from the Golgi apparatus to early enterovirus ROs. A better strategy for direct visualization of ROs in living cells is to label a viral protein involved in their formation. The enterovirus genome can accept coding sequences of foreign proteins (green fluorescent protein [GFP] [27, 28] , proLC3 [16] , Timer [29] , and luciferase [27, 30] among others) at the start of the open reading frame before the capsid coding region. In addition, a poliovirus encoding a viral proteinase (i.e., the 2A protein) tagged with dsRed has been generated (31) . However, 2A is not a bona fide RO marker, as it does not localize exclusively to ROs. Attempts to generate enteroviruses that encode fluorescently labeled, membrane-anchored viral proteins to illuminate ROs have thus far been futile, most likely because fusion of a fluorescent protein to an RO-anchored viral protein impaired its function or liberation from the polyprotein. The use of small genetically encoded tags may overcome this limitation, as evidenced by the successful generation of recombinant polioviruses that encode small epitope tags (such as hemagglutinin [HA] , FLAG, and c-myc) in their 3A protein (32) and a recombinant CVB3 encoding an HA tag in its 2B protein (33) .