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.

Immunofluorescence microscopy. BGM or HeLa cells were grown to subconfluency on coverslips in 24-well plates. Where indicated, cells were transfected with 200 ng of plasmid DNA using Fugene6 according to the manufacturer's protocol or infected with CVB3-3A(S11aa2) at a multiplicity of infection (MOI) of 1 to 10. At 24 h p.t. or 5 to 6 h postinfection (p.i.), cells were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by permeabilization with phosphate-buffered saline (PBS) containing 0.1% Triton X-100 for 10 min. Cells were then incubated sequentially with primary and secondary antibodies diluted in PBS containing 2% normal goat serum. Cellular proteins were detected with primary antibodies against PI4KIII␤ (Millipore), GBF1 (BD Biosciences), and COP-I (provided by F. Wieland, Biochemie-Zentrum, Heidelberg, Germany). GFP(S1-10) was detected with a GFP antibody, and CVB3 3A proteins were detected with a 3A antibody, both described previously (44) . Alexa Fluor 488-or Alexa Fluor 594-conjugated goat anti-rabbit and goat anti-mouse (Molecular Probes) were used as secondary antibodies. Nuclei were stained using 4=,6=-diamidino-2-phenylindole (DAPI). Coverslips were mounted with FluorSave (Calbiochem). Images were acquired with an Olympus BX60 fluorescence microscope with a 40ϫ (0.75-numerical-aperture [NA]) dry objective or a Leica SPE-II DMI-4000 confocal laser scanning microscope equipped with a 100ϫ (1.4-NA) oil immersion objective, and the confocal pinhole was adjusted to 1 airy unit (AU).