JoVE Encyclopedia of Experiments
Immunology
0 views • 4:07 min • July 8th, 2025
Take transfection complexes comprising reporter mRNAs encoding for firefly luciferase, or Fluc, and Renilla luciferase, or Rluc.
Fluc mRNA contains a 5' polyadenosine sequence, or a poly(A) leader, while Rluc mRNA lacks this sequence.
Transfect uninfected and poxvirus-infected mammalian cells to deliver the reporter mRNAs, and incubate.
In uninfected cells, eukaryotic initiation factors bind to the mRNA's 5' cap, followed by recruitment of the small ribosomal subunit and an initiator tRNA.
Upon the small subunit locating the start codon, the large subunit joins to synthesize luciferase proteins.
In infected cells, poxvirus-mediated phosphorylation of the small subunit causes ribosome assembly directly on the leader sequence, enhancing Fluc production.
Lyse the cells to release the luciferases.
Add the Fluc substrate, which the enzyme oxidizes, emitting light. Using a plate reader, measure the luminescence.
Repeat the step for Rluc to determine the relative increase in the Fluc luminescence compared to Rluc in virus-infected cells.
Seed HeLa cells in a 24-well plate to achieve 80% to 90% confluency the following day, and incubate overnight in an incubator at 37 degrees Celsius with
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