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.

Plasmids and infectious clones. The expression construct encoding wild-type (wt) 3A, i.e., p3A-myc, was described elsewhere (64) . GFP(S11), i.e., the residues RDHMVLHEYVNAAGIT, were inserted after amino acid 2 in CVB3 3A to yield p3A(S11aa2)-myc. For this, a forward primer containing the GFP(S11) tag was used in a PCR with p3A-myc as the template to generate 3A(S11aa2) as a PCR product. Wild-type 3A in p3A-myc was replaced with this PCR product using standard DNA cloning techniques. The same strategy was used to introduce GFP(S11) or the StrepII tag (i.e., residues SAWSHPQFEK) in the infectious clone of CVB3 (p53CB3/T7) described elsewhere (64) , yielding p53CB3-3A(S11aa2)/T7 or p53CB3-3A(S-trepIIaa2)/T7. The GFP(S1-10) expression construct pCMV-mGFP(S1-10) (CMV stands for cytomegalovirus, and mGFP stands for modified GFP) was purchased from Sandia Biotech. For the production of CVB3 encoding both GFP(S1-10) and 3A(S11aa2), the GFP(S1-10) gene followed by a 3CD cleavage site was placed directly upstream of the capsid coding region P1 in p53CB3-3A(S11aa2)/T7, similar to modifications previously described for CVB3 encoding luciferase (27) . For the production of murine leukemia virus (MLV) particles, our genes of interest were cloned into the retroviral vectors pQCXIP [for GFP(S1-10) or mCherry-P4M-SidM] or pRetroQ-mCherry (for GM130), both purchased from Clontech, with standard DNA cloning techniques. In the resulting plasmids, the gene of interest is followed by an internal ribosomal entry site (IRES) and a puromycin resistance gene for the production of stable cell lines.