Evolutionary analysis of IFIT1 from clades of mammals reveals rapid evolution.

A. PAML analysis on NCBI-derived coding sequences that were trimmed aligned using CodeML. Likelihood ratio tests were performed to compare model 7 vs model 8 and model 8 vs model 8a to determine the presence of positive selection. N=number of sequences input, and numbers under “positively selected sites” represent residue number of the reference sequence. B. FUBAR analysis on coding sequences that were trimmed and aligned using HyPhy software and DataMonkey. N=number of sequences input, and numbers under “positively selected sites” represent residue number of the reference sequence. C. MEME analysis on coding sequences that were trimmed and aligned using HyPhy software and the DataMonkey application. N=number of sequences input, and numbers under “sites of episodic positive selection” represent residue number of the reference sequence. D. Diagram of Primate IFIT1 domains and location of positively selected sites determined by PAML, FUBAR, and MEME.

Mutagenesis of rapidly evolving residue 193 in human IFIT1 reveals mutational resiliency.

A. Depiction of the human IFIT1 protein with the TPR4 loop location (orange) shown. B. Solved crystal structure of IFIT1 bound to RNA with TPR4 loop forming a “lid” over the exit of the RNA-binding tunnel (PDB:5udj). C. Zoom in on the TPR4 loop and RNA-binding tunnel exit illustrating the location of the TPR4 loop and residue 193 in relation to bound RNA in the human IFIT1 crystal structure. D. (Top) Saturating mutagenesis screen in cells expressing human IFIT1 with residue 193 mutated to every possible residue and challenged with VEEV infection. (Top) (Bottom) Western blot from lysates of Huh7.5 cells expressing IFIT1 point mutants.

IFIT1 ortholog screens reveal extensive heterogeneity in mammalian IFIT1 antiviral function.

A. TimeTree illustrating the evolutionary relationship of IFIT1 orthologs selected for ortholog screen. Scale bar represents divergence time of 10 million years. B. Graph of protein sequence identity of IFIT1 orthologs used in screen relative to human IFIT1. C. Dot plot representing the relative infection (compared to control cells) from an ectopic overexpression screen in which Huh7.5 cells expressing 39 different IFIT1 mammalian orthologs were challenged with 1.0 MOI VEEV-GFP for 4 h. Infectivity was quantified by flow cytometry. Red line denotes 30% relative infection. n=2 biological replicates. D. Same as C, for VSV-GFP (1 MOI, 4 h infection). n=3 biological replicates.

Validation of selected IFIT1s from ortholog screen.

A. TimeTree illustrating the evolutionary relationship of IFIT1 orthologs selected for screen validation and follow-up. Scale bar represents divergence time of 10 million years. B. Western blot from cells expressing HA-tagged IFIT1 orthologs used in C-F. Image is representative of three independent replicates. C. Infection of IFIT1 ortholog-expressing Huh7.5 cells with VEEV-GFP (MOI 2, 4h) D. Infection of IFIT1 ortholog-expressing Huh7.5 cells with VSV-GFP (MOI 2, 4h) E. Infection of IFIT1 ortholog-expressing Huh7.5 cells with PIV3-GFP (MOI 2, 10h) F. Infection of IFIT1 ortholog-expressing Huh7.5 cells with SINV-GFP (MOI 2, 10h). In C-F, data represent mean ± SD, n=3 biological replicates. Statistical significance was determined by one-way ANOVA with Dunnett’s test. ns p > 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

IFIT1 proteins exhibit species-specific Cap0 RNA binding.

A. RNA EMSA with Cap0 VEEV RNA probes (41nt of VEEV TC-83 strain 5’ UTR) at 50nM incubated with increasing concentrations of the indicated purified IFIT1 proteins. Images are one representative image from three independent replicates. B. Plot of the band shift intensity from EMSAs in A. Data represent mean ± SD, n=3. Band intensity was quantified by ImageLab software (BioRad). C. Area Under the Curve (AUC) analysis calculated from raw data in B. Statistical significance was tested by one-way ANOVA with correction for multiple comparisons. Data represent mean ± SD, n=3 replicates. ns, p > 0.05, *, p < 0.05, **, p < 0.01, ***, p < 0.001, and **** p < 0.0001.

Mutagenesis uncovers genetic determinants of primate IFIT1 antiviral function.

A. Diagram and chart describing amino acids that differ between human and chimpanzee IFIT1 as well as their domain location. B. Effects of primate IFIT1 mutant expression on VEEV-GFP infection. Huh7.5 cells were infected with an MOI of 2 for 4 h and infectivity was quantified by flow cytometry. Data represent mean ± SD, n=3 biological replicates. Statistical significance was determined by one-way ANOVA with Dunnett’s test. ns, p > 0.05; ***, p < 0.001; and **** p < 0.0001. Western blotting for HA-tag (IFIT1) and GAPDH (Loading control) was also performed to determine expression levels of IFIT1 mutants. C. ClustalOmega protein sequence alignment of residues 361-367 of IFIT1 from 20 primate species. D. Effects of primate IFIT1 double or triple mutants on VEEV-GFP infection. Huh7.5 cells were infected with an MOI of 2 for 4 h and infectivity was quantified by flow cytometry. Data represent mean ± SD, n=3 biological replicates. Statistical significance was determined by one-way ANOVA with Dunnett’s test. ns, p > 0.05; and **** p < 0.0001. Western blotting for HA-tag (IFIT1) and GAPDH (Loading control) was also performed to determine relative IFIT1 mutant protein abundance. E. Structure of human IFIT1 bound to RNA (left) or AlphaFold predicted structure of chimpanzee IFIT1 (right) visualizing location of residues 364 and 366 (red) that confer antiviral activity. Graphics were generated using ChimeraX. F. Zoom in of (E).

IFIT1 ortholog expression varied during the ortholog screen.

A. Western blot from lysates of cells expressing HA-tagged IFIT1 orthologs for screen in Fig 1A demonstrating unequal IFIT1 expression patterns. Images are representative of three independent replicates. B. Key denoting which symbols correspond to HA-tagged IFIT1 ortholog. Red asterisk marks the size of the truncated or cleaved protein product in lane A3.

Gel image of recombinant IFIT1 protein.

A. Coomassie stained SDS-PAGE gel of purified IFIT1 protein used for RNA EMSA.