Figure 4

αDENV-U143–ΔN Bax constructs effectively target all DENV serotypes A). Ae. albopictus C6/36 cells were transformed with trans-splicing (αDENV-U143) or inactive (αDENV-ΔU143) group I intron vector constructs and maintained under hygromycin selection. U143-ΔN Bax refers to the αDENV-U143 trans-splicing group I intron possessing the pro-apoptotic ΔN Bax as the 3’ exon. ΔU143-ΔN Bax refers to the anti-DENV group I intron (αDENV-ΔU143) possessing the inactive deletion mutation of the trans-splicing domain that is linked to the ΔN Bax 3’ exon. The deletion mutation of the trans-splicing domain is designed to knock out trans-splicing function, providing a negative control [81]. At 15 hours post plating 5x10⁶ cells were infected with A. DENV-1, B. DENV-2, C. DENV-3, D. DENV-4, each at MOI 0.1, and analyzed for the presence of splice product 96 hours p.i. by RT-PCR with heterologous primers. A PCR amplification product derived from a separately constructed spliced sequence control (DNA + Ctrl, see Methods) and a DNA ladder (L) are provided as size standards for each gel. U143-ΔN Bax-D and ΔU143-ΔN Bax-D refer to the active and inactive intron-ΔN Bax constructs respectively that are linked to the DCV –IRES/mCherry configuration as shown in Figure 2. Control RT-PCR experiments were performed with primers for actin to confirm similar RNA loading. Heterolgous primers to the intron- ΔN Bax segment of the construct were used to confirm the presence of our anti-DENV introns. Arrows indicate the predicted size of the principle splice products resulting from intron activity. The identity of spliced products was confirmed by sequencing.