Figure 4. Fiber codon optimization in the adenovirus interferes with the expression of the armed protein when expressed under the control of the Major Late Promoter (MLP).
(A) Schematic representation of the Adwt-EGFP and AdFO-EGFP genomes with the EGFP engineered as an L6 gene under the control of the MLP. (B) EGFP values were analyzed by fluorescence quantification 30 h post-infection in A549 cells infected with Adwt-EGFP or AdFO-EGFP. Left panel shows representative images of EGFP expression. Right panel shows EGFP fluorescence intensity quantification of 20 cells per experiment of three independent experiments shown as a mean ± SEM. (C) Representative western blot of hexon and fiber proteins 30 h post-infection. (D) Intracellular and extracellular viral DNA content analyzed at 30 h post-infection. (E) Comparison of extracellular viral DNA release between AdFO and AdFO-EGFP, versus Adwt and Adwt-EGFP respectively analyzed by qPCR. Cells were infected using 10 TU/cell of both viruses. Data is shown as mean ± SEM of five independent experiments. All AdFO-EGFP fluorescence, intracellular DNA and viral release values are expressed as relative to their corresponding value for Adwt-EGFP in each replicate. ***p < 0.01, ***p < 0.001.
