Extended Data Fig. 6: IFIT2-IFIT3 recognizes short viral 5’ UTRs, related to Fig. 5.
(a-b) 293T cells were transfected with plasmids expressing the 10 nt VSV-P 5’ UTR and GFP with either a short or long plasmid-derived 5’ UTR upstream of the viral sequence, co-transfected in the absence or presence of (a) IFIT2-IFIT3 or (b) IFIT2 or IFIT3 alone. Experiments were performed as described in Fig. 5f-g. (c) Cells were transfected as in panel a, and 22 h post-transfection, cells were harvested, RNA was extracted, and RT-qPCR was conducted with GFP mRNA levels normalized to 18S RNA levels. Data are pooled from two independent experiments, and data are expressed as relative to the short VSV-P UTR alone, no IFIT2-IFIT3 condition. (d) 293T (left), BHK (middle), and H1-Hela (right) cells were transfected with plasmids expressing the 10 nt VSV-P 5’ UTR and GFP in the presence of empty vector (black circles), mouse IFIT2 and IFIT3 (green squares), or human IFIT2 and IFIT3 (orange diamonds). Experiments were performed as described in Fig. 5f-g. (e-f) 293T cells were transfected with plasmids expressing the 10 nt VSV-P 5’ UTR and GFP with either a short (left) or long plasmid-derived (right) 5’ UTR upstream of the viral sequence, co-transfected in the presence of empty vector (black circles), wild-type IFIT2-IFIT3 (green squares), (e) IFIT2-IFIT3 salt-bridge mutants (red triangles) and revertants (green triangles), or (f) IFIT2-IFIT3 RNA binding mutants (red triangles). Experiments were performed as described in Fig. 5g. (g) Cells were transfected with plasmids expressing GFP and the 5’ UTR of each VSV gene in the absence or presence of IFIT2-IFIT3. Experiments were performed as described in Fig. 5 g. Statistical analyses: Data are represented as mean ± SEM. (a, b, c, g) Ordinary two-way ANOVA with Šídák’s multiple comparisons test and a single pooled variance. ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant. (d, e, f) Ordinary one-way ANOVA with Tukey’s multiple comparison test and a single pooled variance. Comparisons are to the empty vector condition within each column. ns = not significant, *** = p < 0.001, **** = p < 0.0001.
