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.

For the delivery of genes [i.e., GFP(S1-10), mCherry-GM130, or mCherry-SidM-P4M] into cells, we used an MLV-based retroviral vector system (Clontech). MLV stocks were produced by cotransfecting HEK293T cells with the pCAGGS-VSV-G plasmid encoding vesicular stomatitis virus protein G (VSV-G), the pMLV-Gag/Pol plasmid encoding the capsid proteins, reverse transcriptase, and integrase proteins, and the plasmid pQCXIP or pRetroQ-mCherry containing the gene of interest. Four to 5 days posttransfection (p.t.), supernatants were harvested and cleared of cell debris by centrifugation and filtration through a 0.45-m-pore-size filter. Stocks were buffered with 10 mM HEPES, aliquoted, and frozen at Ϫ80°C for later use. Prior to transduction, the MLV particles were 5 to 10 times concentrated using a 100-kDa concentrator (Millipore). For the generation of single-cell clones stably expressing GFP(S1-10), BGM and HeLa cells were transduced and grown in the presence of puromycin (2 g/ml for HeLa cells; 30 g/ml for BGM cells) to generate pools of GFP(S1-10)-expressing cells, which were subsequently used to prepare single-cell clones by limiting dilution. Expression of GFP(S1-10) was monitored during passaging by immunofluorescence microscopy using the GFP antibody.