Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to its oligomeric state
- HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, United States
- Cellular Imaging Group, Wellcome Trust Centre for Human Genetics, University of Oxford, United Kingdom
- Department of Infectious Diseases, King’s College London, Faculty of Life Sciences & Medicine, United Kingdom
- Randall Division of Cell and Molecular Biophysics, King’s College London, United Kingdom
Figures
Figure 1 with 1 supplement
Homology-guided identification of a putative oligomerization motif within CD225 domains.
(A) Schematic representation of the membrane topology of IFITM3 made with Protter. Residues corresponding to the amphipathic helix (yellow), palmitoylated cysteines (blue), phenylalanines purported …
Figure 1—figure supplement 1
Quantitative measurement of IFITM3 construct expression following transient transfection and flow cytometric analysis of virus-cell fusion.
(A) HEK293T were transiently transfected with 1.5 µg of empty pQCXIP, IFITM3 WT-FLAG, or the indicated mutants and cells were fixed 48 hr later, stained with anti-FLAG, and assessed by flow …
Figure 2 with 1 supplement
91GxxxG95 is important for restriction of virus entry by IFITM3.
(A) HEK293T cells stably transfected with empty pQCXIP, IFITM3 WT-FLAG, or the indicated mutants were fixed, stained with anti-FLAG antibody and assessed by flow cytometry. FLAG levels are displayed …
Figure 2—figure supplement 1
The subcellular localization of IFITM3 WT and mutants as measured by confocal immunofluorescence microscopy.
(A) HEK293T cells stably expressing IFITM3 constructs were transfected with 0.2 micrograms of EEA1-GFP and cells were fixed at 48 hr post-transfection, permeabilized, and immunostained with …
Figure 3
91GxxxG95 is important for restriction of HIV-1 virion infectivity by IFITM3.
(A) HEK293T were co-transfected with HIV-1 molecular clone pNL4.3 and empty pQCXIP, IFITM3 WT-FLAG, or the indicated mutant. Virus-containing supernatants were harvested at 48 hr post-transfection …
Figure 4 with 2 supplements
91GxxxG95 regulates oligomerization of IFITM3 in living cells.
(A) HEK293T were transiently co-transfected with IFITM3-YFP and mCherry or IFITM3-YFP and IFITM3-mCherry. Constructs encoded IFITM3 WT, IFITM3 G91L, or IFITM3 G95L. FRET-FLIM measurements were made, …
Figure 4—figure supplement 1
Assessment of additional IFITM3 mutations on oligomerization measured by FRET-FLIM.
HEK293T were transiently co-transfected with IFITM3-YFP and mCherry or IFITM3-YFP and IFITM3-mCherry (fluorescent proteins were tagged at the amino-terminus). Constructs encoded IFITM3 WT, IFITM3 …
Figure 4—figure supplement 2
Assessing the functional impact of fluorescent protein placement within IFITM3 fusion proteins.
(A) HEK293T were transiently transfected with 1.5 micrograms of empty pQCXIP, or pQCXIP encoding IFITM3-FLAG (amino-terminal FLAG), IFITM3-(N)YFP (amino-terminal YFP), IFITM3-(N)mCherry …
Figure 5 with 1 supplement
Glycine-95 regulates oligomerization of IFITM3 in denaturing and non-denaturing conditions.
(A) HEK293T were transiently transfected with empty pQCXIP or the following pairs: IFITM3 WT-FLAG and IFITM3 WT-myc or G95L-FLAG and G95L-myc. Whole cell lysates were produced under mildly …
Figure 5—figure supplement 1
Blue native PAGE of IFITM3 and assessment of heteromultimerization between IFITM3 WT-FLAG and G95L-myc.
(A) HEK293T were transiently transfected with empty pQCXIP or the following pairs: IFITM3 WT-FLAG and IFITM3 WT-myc, IFITM3 WT-FLAG and G95L-myc, or G95L-FLAG and G95L-myc. Whole cell lysates were …
Figure 6 with 1 supplement
Membrane order is increased by IFITM3 oligomers in an Ampho B-sensitive manner.
(A) HEK293T cells stably transfected with empty pQCXIP, IFITM3 WT-FLAG, or G95L-FLAG were stained with 1 μM FliptR for 5 min and imaged by FLIM. In the condition indicated, 1 μM Amphotericin B was …
Figure 6—figure supplement 1
Assessment of cholesterol addition and cholesterol depletion on membrane order by FliptR and Laurdan.
(A) Examples of FliptR images from data plotted in Figure 6A. In addition, soluble cholesterol (100 μg/mL) or methyl-beta-cyclo-dextrin (5 mM) were added to untransfected HEK293T cells for 1 hr and …
Figure 7
Disease-associated G305W disrupts the oligomerization of PRRT2 in living cells.
(A) HEK293T were transiently co-transfected with PRRT2-YFP and mCherry or PRRT2-YFP and PRRT2-mCherry. Constructs encoded PRRT2 WT or PRRT2 G305W. Whole-cell FRET analysis was performed on a minimum …
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo-sapiens) | HEK293T | ATCC | CRL-3216 RRID:CVCL_0063 | |
| Cell line (Homo-sapiens) | TZM-bl | NIH AIDS Reagent Resource | 8129–442 RRID:CVCL_B478 | |
| Strain, strain background (Influenza A Virus) | A/PR/8/34 (H1N1) | Charles River Laboratories | 10100781 | Clarified allantoic fluid |
| Recombinant DNA reagent | pNL4-3 (plasmid) | NIH AIDS Reagent Resource | 114 | |
| Recombinant DNA reagent | pNL4-3ΔEnv (plasmid) | Eric O. Freed | ||
| Recombinant DNA reagent | pMD2.G (VSV-G) (plasmid) | Addgene | 12259 RRID:Addgene_12259 | |
| Recombinant DNA reagent | pCMV4-BlaM-Vpr (plasmid) | Addgene | 21950 RRID:Addgene_21950 | |
| Recombinant DNA reagent | pReceiver-YFP-IFITM3 and mutants (plasmid) | This paper | YFP fused to IFITM3 at amino terminus | |
| Recombinant DNA reagent | pReceiver-mCherry-IFITM3 and mutants (plasmid) | This paper | mCherry fused to IFITM3 at amino terminus | |
| Recombinant DNA reagent | pReceiver-PRRT2-YFP and mutants (plasmid) | This paper | YFP fused to PRRT2 at carboxy terminus | |
| Recombinant DNA reagent | pReceiver-PRRT2-mCherry and mutants (plasmid) | This paper | mCherry fused to PRRT2 at carboxy terminus | |
| Recombinant DNA reagent | pQCXIP-FLAG-IFITM3 and mutants (plasmid) | Compton et al., 2014 and this paper | FLAG fused to IFITM3 at amino terminus | |
| Recombinant DNA reagent | EEA1-GFP (plasmid) | Addgene | 42307 RRID:Addgene_42307 | |
| Commercial assay or kit | LiveBLazer FRET-B/G Loading Kit with CCF2-AM | Thermo Fisher | K1032 | |
| Chemicalcompound, drug | Amphotericin B | Sigma | C4951 | |
| Chemical compound, drug | FliptR | Spirochrome | CY-SC020 | |
| Chemical compound, drug | Laurdan | Invitrogen | D250 | |
| Chemical compound, drug | Cholesterol, water-soluble | Sigma | A2942 | |
| Chemical compound, drug | Methyl-beta-cyclo-dextrin | Sigma | C4555 | |
| Antibody | Anti-IAV NP mouse monoclonal | Abcam | AA5H | 1:500 (flow) |
| Antibody | Anti-p24 CA mouse monoclonal | NIH AIDS Reagent Resource | 3537 | 1:1000 (WB) |
| Antibody | Anti-p24 CA [KC57-FITC] mouse monoclonal | BD | CO6604665 | 1:500 (flow) |
| Antibody | Anti-CD63 [MX-49.129.5] mouse monoclonal | Santa Cruz Biotechnology | sc-5275 | 1:400 (IF) |
| Antibody | Anti-FLAG [M2] mouse monoclonal | Sigma | F1804 | 1:1000 (WB) 1:500 (flow) |
| Antibody | Anti-IFITM3 [EPR5242] rabbit monoclonal | Abcam | ab109429 | 1:1000 (WB) 1:200 (IF) |
| Antibody | Anti-Env gp120b sheep polyclonal | NIH AIDS Reagent Resource | 288 | 1:1000 (WB) |
| Antibody | Anti-Env gp41 [2F5] human monoclonal | NIH AIDS Reagent Resource | 1475 | 1:1000 (WB) |
| Antibody | Anti-Actin [C4] mouse monoclonal | Santa Cruz Biotechnology | sc-47778 | 1:1000 (WB) |
| Antibody | Anti-c-Myc rabbit monoclonal | Sigma | C3956 | 1:1000 (WB) |
