Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: Co-precipitation assays between IFIT1 and (A) IFIT2, (B) IFIT5, and (C–D) IFIT3. MBP-tagged IFIT proteins were incubated with amylose resin (lane 1) and unbound MBP-tagged protein was washed (lanes 2–5). Prebound MBP-tagged IFIT (lane 6) was incubated with untagged IFIT proteins (lane 7) prior to washes (lane 8–11) and final beads (lane 12). Bound protein was eluted with maltose-containing buffer (lane 13). Final beads and eluted samples (boxed) were assessed for binding. Data shown are representative of at least two independent experiments. (E) Structure and domain organization of IFIT1 bound to 5′ cap 0 RNA (left), including location of TPR regions. Cartoon representation of IFIT1 colored by subdomain (right). (F) Cartoon representation of IFIT1-FIT3-ap 0 RNA heterotrimeric complex structure: asymmetric unit shows IFIT1 molecule A (green), IFIT1 molecule B (cyan) bound to IFIT3CTD molecule C (magenta), IFIT3CTD molecule D (yellow), and 5′ cap 0 RNA (orange). See also Figure S1 and S2.

Article Snippet: Immunoblots were performed using a rabbit polyclonal anti-IFIT1 (Thermo Fisher), rabbit anti-GAPDH (Cell Signaling Technology), rabbit polyclonal anti-IFIT3 ( Fensterl et al., 2008 ), or mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), followed by IRDye 680RD goat anti-rabbit or IRDye 800CW goat anti-mouse (LiCor).

Techniques: Incubation, Binding Assay

Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: (A) The molecular interface between IFIT1 and IFIT3 is defined by HDX-MS. Differences in deuterium uptake induced by IFIT3 binding are displayed as a color gradient (see legend) and highlighted in the cartoon representation of IFIT1. (B) Comparison of deuterium uptake kinetic curves of IFIT1 (black) and IFIT1-IFIT3 (green). Representative ITC raw data and binding isotherm for (C) IFIT1 and IFIT3. Measured values are KD = 1.5 ± 2.0 nM, ΔH = − 1.3 ± 0.02 × 104 cal/mol, ΔS = − 2.19 cal/mol/deg, and n (no. of sites) = 0.93 ± 0.01. (D) IFIT1 and IFIT3ΔCTD. KD = not determined. (E) IFIT1 and IFIT3CTD. Measured values are KD = 1.4 ± 3.1 nM, ΔH = − 1.7 ± 0.06 × 104 cal/mol, ΔS = − 2.08 cal/mol/deg., and n (number of sites) = 0.97 ± 0.03. (F) IFIT1 and IFIT3CTD K426A-E439A-L445A-S451A-I453A-F457A mutant (IFIT3 CTD mut). KD = not determined. All experiments were performed at least four independent times. See also Figure S3 and S4.

Article Snippet: Immunoblots were performed using a rabbit polyclonal anti-IFIT1 (Thermo Fisher), rabbit anti-GAPDH (Cell Signaling Technology), rabbit polyclonal anti-IFIT3 ( Fensterl et al., 2008 ), or mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), followed by IRDye 680RD goat anti-rabbit or IRDye 800CW goat anti-mouse (LiCor).

Techniques: Binding Assay, Mutagenesis

Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: (A) Comparison of 5′-ppp RNA-bound IFIT5 (green) (PDB 4HOR) and IFIT1 bound to cap 0 RNA and IFIT3CTD (cyan; (primary PDB validation is provided), current structure). (B) Structural changes on IFIT1 upon IFIT3 binding as defined by HDX-MS. Differences in deuterium uptake induced by IFIT3 binding to IFIT1-cap 0 RNA complex are displayed as a color gradient (see legend) and highlighted in the cartoon representation of IFIT1. Data shown are representative of two independent experiments. Multiple peptides were identified by MS/MS, and only the ten highest abundant peptides were used in the analysis. (C) Quantitative filter binding assay for IFIT1 (solid) and IFIT1-IFIT3CTD (dotted) and cap 0 RNA (black), cap1 RNA (red), and 5′-ppp RNA (blue). The results are the average of at least three independent experiments. Error bars represent standard error of the mean (SEM). See also Figure S5.

Article Snippet: Immunoblots were performed using a rabbit polyclonal anti-IFIT1 (Thermo Fisher), rabbit anti-GAPDH (Cell Signaling Technology), rabbit polyclonal anti-IFIT3 ( Fensterl et al., 2008 ), or mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), followed by IRDye 680RD goat anti-rabbit or IRDye 800CW goat anti-mouse (LiCor).

Techniques: Binding Assay, Tandem Mass Spectroscopy, Filter-binding Assay

Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: IFIT3 gene edited (IFIT3mut/mut) 293T cells were trans-complemented with IFIT3 (IFIT3mut/mut + IFIT3), IFIT3 lacking the C-terminal tail (IFIT3mut/mut + IFIT3CTD), or firefly luciferase (IFIT3mut/mut + fluc). (A) 293T-IFIT3mut/mut + IFIT3 and 293T-IFIT3mut/mut + fluc cells were infected at an MOI of 5 with WNV WT or isogenic WNV NS5-E218A lacking 2′-O methylation of the cap structure of genomic RNA. Infection was measured 24 hpi by flow cytometry by staining for intracellular WNV E protein. The fraction of infected 293T-IFIT3mut/mut + IFIT3 cells relative to infected 293T-IFIT3mut/mut + fluc are shown for both viruses. Data are the mean of three independent experiments, and error bars represent SEM. (B–C) 293T-IFIT3mut/mut + IFIT3 and 293T-IFIT3mut/mut + fluc cells were infected with WNV WT or WNV NS5-E218A at an MOI of 0.001. Supernatant was collected at the indicated times after infection and analyzed by focus-forming assay. Data are the mean titers from six independent experiments, and errors bars represent SEM. (D) IFIT3mut/mut + IFIT3, IFIT3mut/mut + hIFIT3ΔCTD, IFIT3mut/mut + mIfit3CTD, and IFIT3mut/mut + fluc cells were stimulated with IFN-β and assessed for IFIT1 expression by Immunoblotting. One representative experiment of three is shown. (E–F) 293T cells expressing human IFIT1-flag under an inducible promoter (293T-IFIT1-doxy) were trans-complemented with IFIT3 or fluc and analyzed for IFIT1 expression (anti-flag tag) by Immunoblotting. (E) One of two representative Immunoblots is shown. (F) IFIT1 amounts were normalized to GAPDH in IFIT3 or fluc-transduced cells treated with doxycycline. Error bars represent SEM from two independent experiments. (G–H) 293T-IFIT1-doxy cells trans-complemented with IFIT3 or fluc were stimulated with doxycycline for 16 h at indicated concentrations, subsequently treated with 50 μM puromycin to arrest translation, and analyzed for IFIT1 (anti-flag tag) at the indicated time post-puromycin treatment. (G) One of three representative Immunoblots is shown. (H) IFIT1 protein amounts were quantified and normalized to the 0 h post-puromycin treatment. Error bars represent SEM from three independent experiments. The statistical significance shown is for the comparison of IFIT3 + 100 ng/ml doxy vs fluc + 100 ng/ml doxy; comparisons between other doses yielded similar results. (I-K) CRISPR-Cas9 gene editing was used to abolish IFIT1 expression from 293T-IFIT3mut/mut + IFIT3 and 293T-IFIT3mut/mut + fluc cells using two different IFIT1-targeting sgRNAs or as a control, scrambled sgRNAs. (I) IFIT1 expression following IFN-β stimulation was determined by Immunoblot. (J–K) 293T-IFIT3mut/mut + IFIT3 and 293T-IFIT3mut/mut + fluc cells that received IFIT1 or scrambled sgRNAs were infected with WNV WT or WNV NS5-E218A at an MOI of 5 (J) or WT or NS5-E218A ZIKV (Cambodia, 2010; strain FSS13025) at an MOI or 1 (K) and scored for infectivity at 24 (J) or 30 (K) hpi by flow cytometry. Infectivity was normalized to the fraction of infected 293T-IFIT3mut/mut + fluc cells that received scrambled sgRNAs. The mean relative infectivity from three (K) and five (J) independent experiments is shown, and error bars represent the SEM. In this Figure, statistical significance was determined using an ANOVA followed by Tukey’s (A, B, and C) or Sidak’s (H, and J–K) multiple comparisons tests (ns, not significant; *, P > 0.05; ***, P <0.001; ****, P < 0.0001). See also Figure S6.

Article Snippet: Immunoblots were performed using a rabbit polyclonal anti-IFIT1 (Thermo Fisher), rabbit anti-GAPDH (Cell Signaling Technology), rabbit polyclonal anti-IFIT3 ( Fensterl et al., 2008 ), or mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), followed by IRDye 680RD goat anti-rabbit or IRDye 800CW goat anti-mouse (LiCor).

Techniques: Luciferase, Infection, Methylation, Flow Cytometry, Staining, Focus Forming Assay, Expressing, Western Blot, FLAG-tag, CRISPR

Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: (A, B and E) 293T-IFIT1-doxy cells were transfected with plasmids encoding fluc, IFIT3, or the indicated IFIT3 mutants, and treated with doxycycline prior to infection with (A) WNV WT or (B and E) WNV NS5 E218A. Infection and transfection data were analyzed by flow cytometry 24 hpi (A and B, left panels; and also see Fig S6E) to determine the percentage of transfected cells that were positive for intracellular WNV E protein. Data are representative of three independent experiments, and error bars represent SEM. (A and B, right panels, and E) Data are normalized to infectivity of doxycycline-untreated cells for each independent transfection (IFIT3, IFIT3 mutants, or fluc) experiment. Errors bars represent the SEM, and data was pooled from three to six independent experiments for statistical analysis (below). (C–D) 293T-IFIT1-doxy cells were trans-complemented with IFIT3 or fluc and infected with (C) VEEV-TC83 (MOI of 1, 16 h) or (D) VSV (MOI of 1, 6 h). Left panels show the mean percentage of infected cells from one representative experiment, and error bars indicate the SEM from triplicate replicates. Right panels show data normalized to infectivity of doxycycline-untreated cells. Errors bars represent the SEM, and data was pooled from three independent experiments. In this Figure, statistical significance was determined using a t-test (A) or an ANOVA followed by Sidak’s (B, C, and D) or Dunnett’s (E) multiple comparisons tests (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). See also Figure S6.

Article Snippet: Immunoblots were performed using a rabbit polyclonal anti-IFIT1 (Thermo Fisher), rabbit anti-GAPDH (Cell Signaling Technology), rabbit polyclonal anti-IFIT3 ( Fensterl et al., 2008 ), or mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), followed by IRDye 680RD goat anti-rabbit or IRDye 800CW goat anti-mouse (LiCor).

Techniques: Transfection, Infection, Flow Cytometry

Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: Co-precipitation assays between IFIT1 and (A) IFIT2, (B) IFIT5, and (C–D) IFIT3. MBP-tagged IFIT proteins were incubated with amylose resin (lane 1) and unbound MBP-tagged protein was washed (lanes 2–5). Prebound MBP-tagged IFIT (lane 6) was incubated with untagged IFIT proteins (lane 7) prior to washes (lane 8–11) and final beads (lane 12). Bound protein was eluted with maltose-containing buffer (lane 13). Final beads and eluted samples (boxed) were assessed for binding. Data shown are representative of at least two independent experiments. (E) Structure and domain organization of IFIT1 bound to 5′ cap 0 RNA (left), including location of TPR regions. Cartoon representation of IFIT1 colored by subdomain (right). (F) Cartoon representation of IFIT1-FIT3-ap 0 RNA heterotrimeric complex structure: asymmetric unit shows IFIT1 molecule A (green), IFIT1 molecule B (cyan) bound to IFIT3CTD molecule C (magenta), IFIT3CTD molecule D (yellow), and 5′ cap 0 RNA (orange). See also Figure S1 and S2.

Article Snippet: Following fixation and permeabilization, cells were co-stained with human WNV-E16, mouse anti-IFIT3 (OTI1G1, Origene), or HA-tagged fluc (mouse anti-HA, R&D Systems).

Techniques: Incubation, Binding Assay

Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: (A) The molecular interface between IFIT1 and IFIT3 is defined by HDX-MS. Differences in deuterium uptake induced by IFIT3 binding are displayed as a color gradient (see legend) and highlighted in the cartoon representation of IFIT1. (B) Comparison of deuterium uptake kinetic curves of IFIT1 (black) and IFIT1-IFIT3 (green). Representative ITC raw data and binding isotherm for (C) IFIT1 and IFIT3. Measured values are KD = 1.5 ± 2.0 nM, ΔH = − 1.3 ± 0.02 × 104 cal/mol, ΔS = − 2.19 cal/mol/deg, and n (no. of sites) = 0.93 ± 0.01. (D) IFIT1 and IFIT3ΔCTD. KD = not determined. (E) IFIT1 and IFIT3CTD. Measured values are KD = 1.4 ± 3.1 nM, ΔH = − 1.7 ± 0.06 × 104 cal/mol, ΔS = − 2.08 cal/mol/deg., and n (number of sites) = 0.97 ± 0.03. (F) IFIT1 and IFIT3CTD K426A-E439A-L445A-S451A-I453A-F457A mutant (IFIT3 CTD mut). KD = not determined. All experiments were performed at least four independent times. See also Figure S3 and S4.

Article Snippet: Following fixation and permeabilization, cells were co-stained with human WNV-E16, mouse anti-IFIT3 (OTI1G1, Origene), or HA-tagged fluc (mouse anti-HA, R&D Systems).

Techniques: Binding Assay, Mutagenesis

Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: (A) Comparison of 5′-ppp RNA-bound IFIT5 (green) (PDB 4HOR) and IFIT1 bound to cap 0 RNA and IFIT3CTD (cyan; (primary PDB validation is provided), current structure). (B) Structural changes on IFIT1 upon IFIT3 binding as defined by HDX-MS. Differences in deuterium uptake induced by IFIT3 binding to IFIT1-cap 0 RNA complex are displayed as a color gradient (see legend) and highlighted in the cartoon representation of IFIT1. Data shown are representative of two independent experiments. Multiple peptides were identified by MS/MS, and only the ten highest abundant peptides were used in the analysis. (C) Quantitative filter binding assay for IFIT1 (solid) and IFIT1-IFIT3CTD (dotted) and cap 0 RNA (black), cap1 RNA (red), and 5′-ppp RNA (blue). The results are the average of at least three independent experiments. Error bars represent standard error of the mean (SEM). See also Figure S5.

Article Snippet: Following fixation and permeabilization, cells were co-stained with human WNV-E16, mouse anti-IFIT3 (OTI1G1, Origene), or HA-tagged fluc (mouse anti-HA, R&D Systems).

Techniques: Binding Assay, Tandem Mass Spectroscopy, Filter-binding Assay

Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: IFIT3 gene edited (IFIT3mut/mut) 293T cells were trans-complemented with IFIT3 (IFIT3mut/mut + IFIT3), IFIT3 lacking the C-terminal tail (IFIT3mut/mut + IFIT3CTD), or firefly luciferase (IFIT3mut/mut + fluc). (A) 293T-IFIT3mut/mut + IFIT3 and 293T-IFIT3mut/mut + fluc cells were infected at an MOI of 5 with WNV WT or isogenic WNV NS5-E218A lacking 2′-O methylation of the cap structure of genomic RNA. Infection was measured 24 hpi by flow cytometry by staining for intracellular WNV E protein. The fraction of infected 293T-IFIT3mut/mut + IFIT3 cells relative to infected 293T-IFIT3mut/mut + fluc are shown for both viruses. Data are the mean of three independent experiments, and error bars represent SEM. (B–C) 293T-IFIT3mut/mut + IFIT3 and 293T-IFIT3mut/mut + fluc cells were infected with WNV WT or WNV NS5-E218A at an MOI of 0.001. Supernatant was collected at the indicated times after infection and analyzed by focus-forming assay. Data are the mean titers from six independent experiments, and errors bars represent SEM. (D) IFIT3mut/mut + IFIT3, IFIT3mut/mut + hIFIT3ΔCTD, IFIT3mut/mut + mIfit3CTD, and IFIT3mut/mut + fluc cells were stimulated with IFN-β and assessed for IFIT1 expression by Immunoblotting. One representative experiment of three is shown. (E–F) 293T cells expressing human IFIT1-flag under an inducible promoter (293T-IFIT1-doxy) were trans-complemented with IFIT3 or fluc and analyzed for IFIT1 expression (anti-flag tag) by Immunoblotting. (E) One of two representative Immunoblots is shown. (F) IFIT1 amounts were normalized to GAPDH in IFIT3 or fluc-transduced cells treated with doxycycline. Error bars represent SEM from two independent experiments. (G–H) 293T-IFIT1-doxy cells trans-complemented with IFIT3 or fluc were stimulated with doxycycline for 16 h at indicated concentrations, subsequently treated with 50 μM puromycin to arrest translation, and analyzed for IFIT1 (anti-flag tag) at the indicated time post-puromycin treatment. (G) One of three representative Immunoblots is shown. (H) IFIT1 protein amounts were quantified and normalized to the 0 h post-puromycin treatment. Error bars represent SEM from three independent experiments. The statistical significance shown is for the comparison of IFIT3 + 100 ng/ml doxy vs fluc + 100 ng/ml doxy; comparisons between other doses yielded similar results. (I-K) CRISPR-Cas9 gene editing was used to abolish IFIT1 expression from 293T-IFIT3mut/mut + IFIT3 and 293T-IFIT3mut/mut + fluc cells using two different IFIT1-targeting sgRNAs or as a control, scrambled sgRNAs. (I) IFIT1 expression following IFN-β stimulation was determined by Immunoblot. (J–K) 293T-IFIT3mut/mut + IFIT3 and 293T-IFIT3mut/mut + fluc cells that received IFIT1 or scrambled sgRNAs were infected with WNV WT or WNV NS5-E218A at an MOI of 5 (J) or WT or NS5-E218A ZIKV (Cambodia, 2010; strain FSS13025) at an MOI or 1 (K) and scored for infectivity at 24 (J) or 30 (K) hpi by flow cytometry. Infectivity was normalized to the fraction of infected 293T-IFIT3mut/mut + fluc cells that received scrambled sgRNAs. The mean relative infectivity from three (K) and five (J) independent experiments is shown, and error bars represent the SEM. In this Figure, statistical significance was determined using an ANOVA followed by Tukey’s (A, B, and C) or Sidak’s (H, and J–K) multiple comparisons tests (ns, not significant; *, P > 0.05; ***, P <0.001; ****, P < 0.0001). See also Figure S6.

Article Snippet: Following fixation and permeabilization, cells were co-stained with human WNV-E16, mouse anti-IFIT3 (OTI1G1, Origene), or HA-tagged fluc (mouse anti-HA, R&D Systems).

Techniques: Luciferase, Infection, Methylation, Flow Cytometry, Staining, Focus Forming Assay, Expressing, Western Blot, FLAG-tag, CRISPR

Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: (A, B and E) 293T-IFIT1-doxy cells were transfected with plasmids encoding fluc, IFIT3, or the indicated IFIT3 mutants, and treated with doxycycline prior to infection with (A) WNV WT or (B and E) WNV NS5 E218A. Infection and transfection data were analyzed by flow cytometry 24 hpi (A and B, left panels; and also see Fig S6E) to determine the percentage of transfected cells that were positive for intracellular WNV E protein. Data are representative of three independent experiments, and error bars represent SEM. (A and B, right panels, and E) Data are normalized to infectivity of doxycycline-untreated cells for each independent transfection (IFIT3, IFIT3 mutants, or fluc) experiment. Errors bars represent the SEM, and data was pooled from three to six independent experiments for statistical analysis (below). (C–D) 293T-IFIT1-doxy cells were trans-complemented with IFIT3 or fluc and infected with (C) VEEV-TC83 (MOI of 1, 16 h) or (D) VSV (MOI of 1, 6 h). Left panels show the mean percentage of infected cells from one representative experiment, and error bars indicate the SEM from triplicate replicates. Right panels show data normalized to infectivity of doxycycline-untreated cells. Errors bars represent the SEM, and data was pooled from three independent experiments. In this Figure, statistical significance was determined using a t-test (A) or an ANOVA followed by Sidak’s (B, C, and D) or Dunnett’s (E) multiple comparisons tests (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). See also Figure S6.

Article Snippet: Following fixation and permeabilization, cells were co-stained with human WNV-E16, mouse anti-IFIT3 (OTI1G1, Origene), or HA-tagged fluc (mouse anti-HA, R&D Systems).

Techniques: Transfection, Infection, Flow Cytometry