Figure 5. Functional analyses of IFIT binding to PPP-RNA.
a, Mobility shift assay between IFIT5 or IFIT3 and ssRNA, dsRNA with blunt ends, or dsRNA with a three-nucleotide (3nt) overhang as indicated by the schematics above each set of lanes. PPP is indicated by red spheres, in vitro transcribed top strand is indicated by the black line, and synthetic complementary RNA is shown in purple. b, Agarose gel-shift assay between IFIT5 and the various RNAs indicated. 7SKas, 7SKantisense; BSA, bovine serum albumin; m7G; 7-methyl-guanosine cap. c, Biotinylated RNA pull-downs (PD) of wild-type (WT) and mutant IFIT1 and IFIT5 from HEK293 cell lysates. QK double indicates Q41E/K150M and QKR triple indicates Q41E/K150M/R253M. Y156F and Y157F were carried out separately, and the appropriate positive and negative controls are described in Supplementary Fig. 17. d, e, PPP-RNA binding is required for antiviral activities of IFIT5 and IFIT1. d, Replication of vesicular stomatitis virus expressing green fluorescent protein (GFP) in doxycycline (Dox)-inducible HEK Flp-In TREx cells expressing IFIT5 (and mutants). Average fold change (± s.d.) in doxycycline-treated versus untreated cells of ten measurements. e, Influenza virus in 293T cells transfected with IFIT1 (and mutants). Average percentage (± s.d.) of influenza polymerase (flu-pol) activity as compared to control (ctrl) of four independent experiments done in duplicate measurements. ***P < 0.001 (one-way analysis of variance, Tukey’s multiple comparison test).