Monitoring Stage-Specific Gene Expression in Vaccinia Virus Using Fluorescent Reporters

Begin with a multiwell plate containing mammalian cells exposed to a recombinant vaccinia virus.

The recombinant virus encodes green, red, and blue fluorescent proteins, each controlled by a stage-specific promoter to track early, intermediate, and late stages of gene expression, respectively.

The virus enters the cell via receptor-mediated endocytosis and releases its viral core into the cytoplasm.

The transcriptional machinery in the viral core drives early gene expression, marked by green fluorescence.

Early proteins mediate genome uncoating, releasing viral DNA into the cytoplasm, where DNA polymerase replicates the viral genome.

Next, intermediate gene expression, as indicated by red fluorescence, produces transcription factors essential for activating late genes.

Then, late gene expression, as indicated by blue fluorescence, generates structural proteins that are necessary for forming mature virions.

Add a fixative and incubate briefly to preserve fluorescence.

Remove the fixative, then add a buffer.

Use a spectrophotometer to measure fluorescence and quantify stage-specific viral gene expression in infected cells.

Begin this protocol by dissociating Hela cells grown on a 10 centimeter