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.

CVB3 that encodes not only 3A(S11aa2) but also GFP(S1-10) (Fig. 7A) . To do this, the gene encoding GFP(S1-10) was inserted upstream of the capsid coding region (P1) in the infectious clone containing 3A(S11aa2). GFP(S1-10) was followed by an artificial 3CD cleavage site to release the protein from the P1 region upon translation. Viable virus [i.e., CVB3-GFP(S1-10)-3A(S11aa2)] was obtained upon transfection of BGM cells with RNA transcripts of the infectious clone. After harvesting CVB3-GFP(S1-10)-3A(S11aa2), we compared its replication kinetics in BGM cells to CVB3-3A(S11aa2) in BGM(GFPS1-10) cells, so that in both cases the 3A(S11aa2) protein would bind to GFP(S1-10). The levels of viral RNA replication were nearly identical for both viruses (Fig. 7B) , suggesting that the generation of ROs during the course of infection occurs similarly. However, the virus encoding both GFP fragments [i.e., CVB3-GFP(S1-10)-3A(S11aa2)] exhibited delayed production of infectious progeny compared to CVB3-3A(S11aa2) (Fig. 7B) . In line with a previous study (51) , we found that the processing of the artificial 3CD cleavage site between a foreign protein and P1 is suboptimal (see Fig. S3 in the supplemental material), which may explain the delay in progeny virion production. FIG 7 Construction of CVB3 that encodes both GFP(S1-10) and 3A(S11aa2). (A) Schematic diagram of the genome organization of CVB3 showing the insertion of GFP(S1-10) before the capsid coding region and GFP(S11) in 3A after amino acid 2. 5= UTR, 5= untranslated region. (B) Growth curve analysis of CVB3-GFP(S1-10)-3A(S11aa2) in BGM cells and CVB3-3A(S11aa2) in BGM(GFPS1-10) cells. Cells were infected for 30 min at an MOI of 1. At the indicated time points, cells were subjected to titration analysis after freeze-thawing cycles to determine the amount of infectious virus particles. Alternatively, cells were lysed to determine the amount of viral RNA by quantitative PCR. The results are expressed as fold induction relative to the quantities determined directly after removing the inoculum. (C) BGM cells were infected with CVB3 GFP(S1-10) 3A(S11aa2). At 5 and 6 h p.i., cells were fixed and subjected to immunofluorescence analysis. The detected GFP fluorescence resulted from the assembly of GFP(S1-10) with 3A(S11aa2).