IFIT1 is rapidly evolving and exhibits disparate antiviral activities across 11 mammalian orders
- Department of Microbiology, University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 1 supplement
Evolutionary analysis of Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) from clades of mammals reveals rapid evolution.
(A) Phylogenetic Analysis using Maximum Likelihood (PAML) analysis on NCBI-derived coding sequences that were aligned and then analyzed using CodeML. Likelihood ratio tests were performed to compare model 7 vs model 8 and model 8 vs model 8 a 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) Fast Unconstrained Bayesian AppRoximation (FUBAR) analysis on aligned coding sequences using HyPhy software and the DataMonkey application. N=number of sequences input, and numbers under ‘positively selected sites’ represent residue number of the reference sequence. (C) Mixed Effects Model of Evolution (MEME) analysis on aligned coding sequences 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.
Figure 1—figure supplement 1
Analysis of Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) for recombination and positive selection.
(A) Maximum likelihood tree of C-terminal region of IFIT1, IFIT1B, and IFIT3 coding sequences (corresponding to bases 907–1437 of human IFIT1 coding sequence), generated in MEGA X with 1000 bootstrapping replicates. Select bootstrap values are shown. (B) GARD analysis of IFIT1 coding sequences used for evolutionary analysis in Figure 1. (C) Phylogenetic Analysis using Maximum Likelihood (PAML) analysis on regions of IFIT1 coding sequence after (primate and ungulate) or between (bat and carnivore) breakpoints indicated in B. (D) Fast Unconstrained Bayesian AppRoximation (FUBAR) analysis on regions of IFIT1 coding sequence after (primate and ungulate) or between (bat and carnivore) breakpoints indicated in B. (E) Mixed Effects Model of Evolution (MEME) analysis on regions of IFIT1 coding sequence after (primate and ungulate) or between (bat and carnivore) breakpoints indicated in B. (F) PAML, FUBAR, and MEME analysis on primate IFIT1 coding sequence after removal of inferred recombinant sequences by Recombination Detection Program 4 (RDP4).
Figure 2
Mutagenesis of rapidly evolving residue 193 in human Interferon-induced protein with tetratricopeptide repeats 1 (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 Venezuelan equine encephalitis virus (VEEV) infection. Data represent mean +/- SD, n=3 independent experiments. (Bottom) Western blot from lysates of Huh7.5 cells expressing IFIT1 point mutants.
-
Figure 2—source data 1
Original images of membranes corresponding to Figure 2, panel D.
Molecular weight was determined by PageRuler Plus Prestained Protein Ladder (Thermo Scientific).
- https://cdn.elifesciences.org/articles/101929/elife-101929-fig2-data1-v1.zip
-
Figure 2—source data 2
Original files for western blot analysis displayed in Figure 2.
- https://cdn.elifesciences.org/articles/101929/elife-101929-fig2-data2-v1.zip
Figure 3 with 1 supplement
Interferon-induced protein with tetratricopeptide repeats 1 (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 hr. Infectivity was quantified by flow cytometry. Red line denotes 30% relative infection. n=2 independent experiments. (D) Same as C, for VSV-GFP (1 MOI, 4 hr infection). n=3 independent experiments. In C, D, silhouettes represent the species with a relative inhibition of at least 70% in order from most (top) to least (bottom) inhibitory.
Figure 3—figure supplement 1
Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) ortholog expression varied during the ortholog screen.
(A) Western blot from lysates of cells expressing HA-tagged IFIT1 orthologs for screen in Figure 3 demonstrating unequal IFIT1 expression patterns. Images are representative of three independent experiments. (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.
-
Figure 3—figure supplement 1—source data 1
Original images of membranes corresponding to Figure 3—figure supplement 1A.
Molecular weight was determined by PageRuler Plus Prestained Protein Ladder (Thermo Scientific).
- https://cdn.elifesciences.org/articles/101929/elife-101929-fig3-figsupp1-data1-v1.zip
-
Figure 3—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/101929/elife-101929-fig3-figsupp1-data2-v1.zip
Figure 4
Validation of selected Interferon-induced protein with tetratricopeptide repeats 1 (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. Quantification of band intensity was performed using LiCOR Image Studio C. Infection of IFIT1 ortholog-expressing Huh7.5 cells with VEEV-GFP (MOI 2, 4 hr) (D) Infection of IFIT1 ortholog-expressing Huh7.5 cells with VSV-GFP (MOI 2, 4 hr). (E) Infection of IFIT1 ortholog-expressing Huh7.5 cells with PIV3-GFP (MOI 2, 10 hr). (F) Infection of IFIT1 ortholog-expressing Huh7.5 cells with SINV-GFP (MOI 2, 10 hr). (G) Heat map summarizing the infection data shown in C-F and complementary ortholog expression data shown in B. In C-F, data represent mean ± SD, n=3 independent experiments. 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.
-
Figure 4—source data 1
Original images of membranes corresponding to Figure 4B.
Molecular weight was determined by PageRuler Plus Prestained Protein Ladder (Thermo Scientific).
- https://cdn.elifesciences.org/articles/101929/elife-101929-fig4-data1-v1.zip
-
Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4B.
- https://cdn.elifesciences.org/articles/101929/elife-101929-fig4-data2-v1.zip
Figure 5 with 1 supplement
Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) proteins exhibit species-specific Cap0 RNA binding.
(A) RNA electrophoretic mobility shift assay (EMSA) with Cap0 Venezuelan equine encephalitis virus (VEEV) RNA probes (41 nt of VEEV TC-83 strain 5’ UTR) at 50 nM 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 independent experiments. 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 independent experiments. ns, p>0.05, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
-
Figure 5—source data 1
Original images of SYBR Gold-stained native gels corresponding to Figure 5A.
- https://cdn.elifesciences.org/articles/101929/elife-101929-fig5-data1-v1.zip
-
Figure 5—source data 2
Original files for electrophoretic mobility shift assay (EMSA) displayed in Figure 5A.
- https://cdn.elifesciences.org/articles/101929/elife-101929-fig5-data2-v1.zip
Figure 5—figure supplement 1
Gel image of recombinant Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) protein.
(A) Coomassie-stained SDS-PAGE gel of purified IFIT1 protein used for RNA electrophoretic mobility shift assay (EMSA).
-
Figure 5—figure supplement 1—source data 1
Original image of Coomassie-stained SDS-PAGE gel corresponding to Figure 5—figure supplement 1.
Molecular weight was determined by PageRuler Plus Prestained Protein Ladder (Thermo Scientific).
- https://cdn.elifesciences.org/articles/101929/elife-101929-fig5-figsupp1-data1-v1.zip
-
Figure 5—figure supplement 1—source data 2
Original file for SDS-PAGE gel displayed in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/101929/elife-101929-fig5-figsupp1-data2-v1.zip
Figure 6
Mutagenesis uncovers genetic determinants of primate Interferon-induced protein with tetratricopeptide repeats 1 (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 Venezuelan equine encephalitis virus (VEEV)-GFP infection. Huh7.5 cells were infected with an MOI of 2 for 4 hr and infectivity was quantified by flow cytometry. Data represent mean ± SD, n=3 independent experiments. 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) Clustal Omega 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 hr and infectivity was quantified by flow cytometry. Data represent mean ± SD, n=3 independent experiments. 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).
-
Figure 6—source data 1
Original images of membranes corresponding to Figure 6B, (top), and Figure 6D, (bottom).
Molecular weight was determined by PageRuler Plus Prestained Protein Ladder (Thermo Scientific).
- https://cdn.elifesciences.org/articles/101929/elife-101929-fig6-data1-v1.zip
-
Figure 6—source data 2
Original files for western blot analysis displayed in Figure 6.
- https://cdn.elifesciences.org/articles/101929/elife-101929-fig6-data2-v1.zip
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/101929/elife-101929-mdarchecklist1-v1.docx
-
Supplementary file 1
IFIT1 ortholog screen sequence information and viral infection data.
- https://cdn.elifesciences.org/articles/101929/elife-101929-supp1-v1.xlsx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
IFIT1 is rapidly evolving and exhibits disparate antiviral activities across 11 mammalian orders
eLife 13:RP101929.
https://doi.org/10.7554/eLife.101929.3
