FIG 3 .
Recombinant SeV iDVGs with an intact DVG70-114 motif preserve their stimulatory activity in the context of infection. (A) Schematic of the recombinant SeV DVG generation system. BSR-T7 cells were infected with partially inactivated SeV 52 and transfected with a plasmid encoding either DVG-324 or DVG-354. Cells and supernatants were collected 48 h later and inoculated into 10-day-old embryonated chicken eggs. SeV containing rDVGs was collected from the allantoic fluid. T7 pro, T7 promoter sequence; Riboz., ribozyme; T7 term, T7 polymerase terminator sequence. (B) LL-CMK2 cells were infected with virus rDVG-324 or rDVG-354 at an MOI of 5 TCID50/cell and analyzed at 6 h postinfection by RT-qPCR for the expression of IFNB1 and IFIT1 mRNA, SeV NP mRNA, and DVGs. The relative copy number of DVG RNA was quantified by RT-qPCR with the DVG comp primers (see Table S1 in the supplemental material). The data are the average of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant (one-way ANOVA with Bonferroni’s post hoc test and two-tailed t test in DVG RNA quantification). Data are expressed as the copy number relative to that of the housekeeping gene for GAPDH. (C and D) LL-CMK2 cells were infected with SeV LD at an MOI of 1.5 TCID50/cell and transfected 24 h later with 4.15 pmol of DVG-268 or DVG-268Δ70-114 RNA or left untreated (NT). Expression of SeV NP and SeV (P/V) (C) and antiviral genes (D) was measured at 6 h posttransfection. The data are the average of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant (one-way ANOVA with Bonferroni’s post hoc test). Data are expressed as the copy number relative to that of the housekeeping gene for GAPDH.