Figure 1. HeLa cells infected with Timer-CVB3 slowly change fluorescence from green to red.

The gene for “fluorescent timer” protein was inserted into the infectious plasmid clone for CVB3 (pMKS1) which contains a unique Sfi1 restriction site followed by the coding sequence for a polyglycine linker and a 3C^pro/3CD^pro cleavage site (shown as a purple circle) immediately downstream of the viral polyprotein start codon. Upon HeLa cell infection with recombinant CVB3 expressing “fluorescent timer” protein (Timer-CVB3), we expected the slow conversion of the green fluorescing form of timer protein to red (shown diagrammatically). HeLa cells infected with Timer-CVB3 (moi = 0.1) initially fluoresced green (recent viral protein) at 24 hours PI as determined by fluorescence microscopy. By 48 hours PI, both green and red fluorescence (matured viral protein) was observed in infected HeLa cells, although the majority of cells fluoresced brightly in the red channel.