Fig. 5: IFIT2–IFIT3 recognizes short viral 5’ UTRs.
a, The 5’ UTR and ORF of each VSV gene was cloned into a mammalian expression plasmid fused to a C-terminal V5 tag (schematic at top). On the basis of 5’ RACE data (Extended Data Fig. 5a), we identified a 108-nt plasmid-derived extension (grey) appended to the 5’ end of the cloned VSV 5’ UTR (black). Each viral gene-expressing construct was co-transfected in the absence or presence of IFIT2–IFIT3 into HEK293T cells. At 24 h post transfection, cells were collected and lysates were analysed by western blotting. b, Following plasmid engineering, only 3 nt of plasmid-derived sequence remained at the 5’ end of each VSV UTR and ORF (schematic at top; Extended Data Fig. 5b). Transfections and western blotting were performed as in a. In a and b, experiments were performed independently at least twice. c, 5’ UTR and ORF schematics for fluorescence reporter constructs shown in d and e. d, HEK293T cells were transfected with the indicated GFP reporters in the absence or presence of co-transfected IFIT2–IFIT3. All wells were transfected with the control mCherry normalization construct. Images were taken at 24 h post transfection. Scale bars, 200 μm. e, Experiments were performed as in d. For each well (n = 4 biological replicates), 4 images were taken and the ratio of GFP:mCherry signal intensity (Extended Data Fig. 5d) was calculated for each image. All values were normalized to the average of the condition in which IFIT2–IFIT3 was not transfected. f, 5’ UTR and ORF schematics for GFP reporter constructs shown in g. AA, amino acids. g, Experiments with the indicated constructs were performed and quantified as in e. h, 5’ UTR and ORF schematics for GFP reporter constructs shown in i. Plasmid-derived sequence from the ‘long’ construct shown in c was added back with the indicated number of nt (for example, Plus 10). i, Experiments with the indicated constructs were performed and quantified as in e (n = 10 biological replicates). For e, g and i, data are represented as mean ± s.e.m. Statistical analyses: ordinary two-way ANOVA with Šídák’s post test and a single pooled variance. ****P < 0.0001, ***P = 0.0002.
