Natural mutations in IFITM3 modulate post-translational regulation and toggle antiviral specificity

Abstract

The interferon-induced transmembrane (IFITM) proteins protect host cells from diverse virus infections. IFITM proteins also incorporate into HIV-1 virions and inhibit virus fusion and cell-to-cell spread, with IFITM3 showing the greatest potency. Here, we report that amino-terminal mutants of IFITM3 preventing ubiquitination and endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV-1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino-terminal mutations that modify protein localization and function. This suggests that "runaway" IFITM3 variants could be selected for altered antiviral activity. Furthermore, we show that adaptations in IFITM3 result in a trade-off in antiviral specificity, as variants exhibiting enhanced activity against HIV-1 poorly restrict influenza A virus. Overall, we provide the first experimental evidence that diversification of IFITM3 genes may boost the antiviral coverage of host cells and provide selective functional advantages.

Keywords: HIV; IFITM; evolution; innate immunity; virus.

Figure 1. Mutation in IFITM3 alters protein subcellular localization and anti‐HIV activity

1. A schematic of the IFITM3 protein is shown, with gray boxes indicating transmembrane domains and colored bars indicating residues targeted for post‐translational modification: ubiquitinated lysines (blue), palmitoylated cysteines (purple), and a phosphorylated tyrosine (red). Residues 17–24 are shown in detail to highlight two adjacent, overlapping motifs that recruit NEDD4 (PPNY) and the AP‐2 complex (YEML). The IFITM3 Δ1–21 variant initiates translation at an internal methionine residue (green) and thus lacks these motifs.

2. Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP encoding IFITM3 variants containing N‐terminal FLAG (pQCXIP‐FLAG‐IFITM3) and immunostained with anti‐FLAG M2 antibody. Scale bar, 5 μm.

3. Flow cytometric analysis of transfected cells in (B) immunostained with anti‐FLAG M2 antibody. Corresponding mean fluorescence intensity values are shown.

4. SDS–PAGE of cell lysates derived from 293T cells transfected in (B) followed by immunoblotting with anti‐FLAG M2 and anti‐actin antibodies.

5. SDS–PAGE of OptiPrep‐purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP‐FLAG‐IFITM3 variants, HIV‐1 pNL4‐3, and Vpr‐Blam plasmid followed by immunoblotting with anti‐FLAG M2 and anti‐p24 Gag (183‐H12‐5C).

6. OptiPrep‐purified supernatants from (E) were incubated with SupT1 target cells for 4 h and virus–cell fusion was scored by flow cytometry. Mean + SD of 5–10 experiments (each performed with virus produced from an independent transfection) is shown.

7. Viruses used in (E) were used to infect SupT1 cells and productive infection was scored at 48–60 h by immunostaining with the KC57 antibody and flow cytometry. Expression of pCMV‐MxA served as a negative control. The mean + SD of 5–10 experiments is shown.

8. Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP‐FLAG‐IFITM3 variants and immunostained with anti‐FLAG M2 antibody.

9. SDS–PAGE of cell lysates derived from 293T cells transfected in (H) followed by immunoblotting with anti‐FLAG M2 and anti‐actin antibodies. Scale bar, 5 μm.

10. SDS–PAGE of OptiPrep‐purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP‐FLAG‐IFITM3 constructs, HIV‐1 pNL4‐3, and Vpr‐Blam plasmid followed by immunoblotting with anti‐FLAG M2 and anti‐p24 Gag.

11. OptiPrep‐purified supernatants from (J) were incubated with SupT1 target cells for 4 h and virus–cell fusion was scored by flow cytometry. The mean + SD of three experiments is shown.

Data information: Western blot and microscopy images are representative of three independent experiments. Unpaired t‐test (two‐sided), **P < 0.005. Statistical comparisons were made between the condition indicated and IFITM3 WT. Normality was determined by the KS test and variance determined by the F‐test.

Figure EV1. Dissection of two negative regulatory motifs in IFITM3 reveals additive roles in anti‐HIV activity

1. Flow cytometric analysis of IFITM3 protein levels in 293T, HeLa, Tet‐ON SupT1, and primary CD4⁺ T cells. Staining was performed with the mouse monoclonal anti‐IFITM3 antibody (Proteintech, 66081‐1‐Ig) with or without stimulation by type‐I IFN (1,000 U/ml) or doxycycline for 24 h.

2. Confocal fluorescence microscopy of 293T cells transfected with 1 μg pNL4‐3 and 0.5 μg pQCXIP‐FLAG‐IFITM3 or pQCXIP‐FLAG‐IFITM3 Δ1–21. Immunostaining was performed with anti‐FLAG M2 and anti‐Gag p17 (ARP342). Scale bars, 5 μm.

3. Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP‐FLAG‐IFITM3 ΔY20 or pCMV‐myc‐IFITM3 L23Q and immunostained with anti‐FLAG M2 or anti‐IFITM3 (Proteintech, 66081‐1‐Ig), respectively.

4. SDS–PAGE of OptiPrep‐purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP‐FLAG‐IFITM3 or pQCXIP‐FLAG‐IFITM3 ΔY20, HIV‐1 pNL4‐3, and Vpr‐Blam plasmid followed by immunoblotting with anti‐FLAG M2 and anti‐p24 Gag.

5. Virus supernatants from (D) were incubated with SupT1 target cells for 4 h and virus–cell fusion was scored by flow cytometry. Mean + SD of three experiments is shown.

6. Flow cytometric analysis of 293T cells transfected with 0.5 μg of pQCXIP‐FLAG‐IFITM3 constructs and immunostained with anti‐FLAG M2 antibody. Corresponding mean fluorescence intensity values are shown.

7. Virus produced in Fig 1K was used to infect SupT1 cells and productive infection was scored at 48–60 h by immunostaining with the KC57 antibody and flow cytometry. Mean + SD of three experiments is shown.

8. OptiPrep‐purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pCMV‐myc‐IFITM3 or pCMV‐myc‐IFITM3 L23Q or pCMV‐MxA constructs, HIV‐1 pNL4‐3, and Vpr‐Blam plasmid were incubated with SupT1 target cells for 4 h and virus–cell fusion was scored by flow cytometry.

9. Flow cytometric analysis of 293T cells transfected with 0.5 μg of pCMV constructs and immunostained with anti‐IFITM3 or anti‐MxA antibodies. For these experiments, IFITM3 and the L23Q mutant were expressed from a different plasmid background (pCMV) and protein levels and antiviral activity were generally increased. Of note, the L23Q mutant did not achieve higher relative protein expression, suggesting that its enhancement at the cell surface is responsible for its superior anti‐HIV activity.

Data information: Western blot and microscopy images are representative of three independent experiments. Unpaired t‐test, *P < 0.05, **P < 0.005.

Figure EV2. The E3 ubiquitin ligase NEDD4 regulates IFITM3 in HIV‐1‐infected cells

1. SDS–PAGE of cell lysates (upper) and OptiPrep‐purified virus (lower) derived from 293T cells transfected with 0.2 μg of pQCXIP‐FLAG‐IFITM3 or pQCXIP‐FLAG‐IFITM3 Δ1–21, 1.0 μg of HIV‐1 pNL4‐3, and 0.5 μg pCI‐HA‐NEDD4 WT or pCI‐HA‐NEDD4 C867A plasmids. Immunoblotting was performed with anti‐NEDD4, anti‐FLAG M2, and anti‐actin on whole‐cell lysates, while anti‐FLAG M2 and anti‐p24 Gag were used on 15 ng p24 equivalents of purified virus.

2. Confocal fluorescence microscopy of 293T cells transfected with pQCXIP‐FLAG‐IFITM3 and pCI‐HA‐NEDD4 or pCI‐HA‐NEDD4 C867A or pCI‐Empty, followed by immunostaining with anti‐FLAG M2 and anti‐HA. A single medial Z slice is shown.

3. A second example of cells from (B) in which pQCXIP‐FLAG‐IFITM3 and pCI‐HA‐NEDD4 are co‐transfected. A single medial Z slice (upper) and a 3D reconstruction of Z‐stacks (lower) are shown.

4. 293T cells were transfected with pQCXIP‐FLAG‐IFITM3 or pQCXIP‐FLAG‐IFITM3 Δ1–21 and pCI‐HA‐NEDD4, and immunostaining was performed with anti‐FLAG M2 and anti‐LAMP1.

Data information: Western blot and microscopy images are representative of three independent experiments. Scale bars, 5 μm in (B) and (C) and 10 μm in (D).

Figure EV3. Assessment of virion composition by supernatant fractionation

Virus‐containing supernatants produced from Tet‐ON IFITM3⁺ or Δ1–21⁺ cells were fractionated using a 6–18% OptiPrep gradient velocity gradient. Fractions were analyzed by SDS–PAGE and immunoblotted with anti‐p24 Gag and anti‐FLAG M2 antibodies. Western blot images are representative of two independent experiments.

Figure 2. Mutation of N‐terminal regulatory motifs of IFITM3 in T cells enhances the restriction of HIV‐1

1. Confocal fluorescence microscopy of SupT1 T cells transduced with Tet‐inducible pQCXIP‐FLAG‐IFITM3 or pQCXIP‐FLAG‐IFITM3 Δ1–21 (Tet‐ON) immunostained with anti‐FLAG M2 antibody following overnight treatment with 500 ng/ml doxycycline. Scale bar, 10 μm.

2. Tet‐ON SupT1 cell lines were treated or not with 500 ng/ml doxycycline overnight and induction of IFITM3 protein was assessed by anti‐FLAG M2 immunostaining and flow cytometry.

3. Tet‐ON SupT1 cell lines were productively infected with NL4‐3 VSV‐G and then treated with 500 ng/ml doxycycline overnight to produce virus from IFITM3⁻ and IFITM3⁺ cells; 25 ng p24 equivalents of purified supernatants was used to infect fresh Tet‐ON SupT1 cells, which were previously treated with 500 ng/ml doxycycline or not. Infection was scored at 72 h postinfection by immunostaining with KC57 and flow cytometry. Mean + SD of three experiments is shown.

4. About 25 ng p24 equivalents of virus used in (C) was subjected to SDS–PAGE. Immunoblotting was performed using anti‐FLAG M2, anti‐p24 Gag, and anti‐gp120 Env (NIH #288) antibodies.

5. 293T cells were transfected with the indicated amount of pQCXIP‐FLAG‐IFITM3 or pQCXIP‐FLAG‐Δ1–21 or pCMV‐MxA and 1.0 μg of HIV‐1 pNL4‐3. SDS–PAGE was performed on whole‐cell lysates followed by immunoblotting with anti‐FLAG M2, anti‐p24 Gag, anti‐Env gp120 (NIH #288), and anti‐tubulin antibodies.

Data information: Western blot and microscopy images are representative of three independent experiments. Unpaired t‐test, *P < 0.05, ^♯ P = 0.12, nonstatistically significant. Comparisons were made between the condition indicated and the corresponding condition in Tet‐ON IFITM3 cells.

Figure 3. Retracing the origins of IFITM in primates

1. A comparison of the IFITM locus on chromosome 11 of the human reference genome (GRCh38.p5) and the corresponding locus in the bushbaby reference genome (OtoGar3) visualized using Genomicus v84.01. Synteny is observed between human IFITM and bushbaby IFITM, as evidenced by the conserved flanking genes B4GALNT4 and ATHL1. The bushbaby locus lacks IFITM2 and IFITM1 genes. In addition, it contains two adjacent IFITM3 genes, as well as a third IFITM3 gene located in a different genomic context, indicative of gene duplication events.

2. A gene gain/loss tree summarizing the phylogenetic history of the IFITM gene family in primates, using annotated reference genomes in Ensembl. The number at a branch tip refers to the number of IFITM genes present in a given extant species, while the number at each node refers to the number present in an ancestral species. Differences in gene number at tips and nodes represent significant gene gain events (expansions) or gene loss events (contractions) based on a P‐value calculation of < 0.01 determined by CAFE (Computational Analysis of gene Family Evolution) 55. The makeup of the IFITM repertoire for each species, as determined by comparative genomics and the IFITM gene family tree in Ensembl, is detailed to the right. When multiple copies of IFITM genes were identified, the total number is listed inside the box. The gene numbers estimated for the mouse lemur and bushbaby in Ensembl were adjusted following phylogenetic analysis of IFITM homologs in Genomicus as well as BLAST searches of the NCBI GenBank database. Blue and red arrows indicate the emergence of IFITM1 and IFITM2 genes, respectively.

Figure 4. Recurrent duplication and divergence of primate IFITM3 over evolutionary time

1. A partial protein alignment of select IFITM3 genes from various primate species for which a reference genome is available and accessible in Genomicus. Sequences from the mouse lemur and colobus monkey were identified by BLAST analysis of reference genomes in NCBI GenBank. Gene location is indicated, when available, by chromosome number, and an asterisk indicates that the given gene sequence falls into a canonical IFITM locus with flanking B4GALNT4 and ATHL1 genes. The suborder and parvorder (from left to right) indicate taxonomical relationships between species. Residues bolded in red highlight nonsynonymous substitutions that disrupt canonical motifs for the recruitment of NEDD4 and AP‐2. A complete protein alignment of all IFITM sequences analyzed in this study is available in Appendix.

2. A phylogenetic gene tree (cladogram) indicates the relationship among IFITM3 sequences analyzed in this study. The final consensus tree was made by merging results from neighbor joining and maximum‐likelihood phylogenetic reconstructions using both nucleotide and amino acid sequences in Ensembl (see Fig EV4 for robustness measurement by Bayesian inference). Branch lengths are normalized and arbitrary. The size of triangles at the tree tips roughly corresponds to the number of sequences contained therein. Note that the IFITM2 gene is the most recent member of the IFITM locus, appearing only in humans, chimpanzee, and gorilla genomes.

3. A graphical representation of naturally occurring truncated forms of IFITM3 identified in the vervet and marmoset reference genomes, as compared to the putative Δ1–21 variant in humans.

Figure EV4. A Bayesian phylogenetic reconstruction of primate IFITM3 and IFITM2

1. A phylogenetic tree based on select IFITM3 and IFITM2 genes aligned pairwise using T‐Coffee. Ambiguous regions, including gaps, were resolved with Gblocks. The tree was reconstructed using the Bayesian inference method implemented in the MrBayes program (v3.2.3). The number of substitution types was fixed to 6. The standard (4 × 4) model of nucleotide substitutions was used, with rates variation across sites fixed to “invariable + gamma”. Four Markov chain Monte Carlo (MCMC) chains were run for 100,000 generations, with sampling every 10 generations and the first 500 sampled trees discarded as “burn‐in”. A 50% majority rule was used for the placement of ancestral nodes. Branch annotations are Bayesian posterior probabilities used as a measure of tree robustness. Sequences in bold are those discussed in Fig 4 and functionally characterized in Fig 5.

2. The full proteins of putative human IFITM3 variant Δ1–21, marmoset IFITM3 ENSCJAG00000037392, and vervet IFITM3 ENSCSAG00000019450 were aligned via Clustal Omega.

Figure 5. Natural mutations in IFITM3 disrupt regulatory motifs and toggle antiviral activity

1. Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg of pQCXIP‐FLAG‐IFITM3 mutated at the indicated residues and immunostained with anti‐FLAG M2 antibody. Scale bars, 5 μm. The P17L, P18H, Δ22–24, P18L/L23P, and P18H/Δ19–23 mutations were introduced into the human IFITM3 background.

2. SDS–PAGE of FLAG‐immunoprecipitated fractions from 293T cells following transfection of 0.5 μg of pQCXIP‐FLAG‐IFITM3 variants and 0.5 μg of pCI‐HA‐NEDD4 or pCI‐HA‐NEDD4 C867A. Immunoblotting was performed with anti‐FLAG M2, anti‐IFITM3 (Abcam, EPR5242), and an anti‐ubiquitin antibody (Enzo Life Sciences, recognizing K²⁹‐, K⁴⁸‐, and K⁶³‐linked mono‐ and polyubiquitinylated proteins). “L” indicates the presence of light chain antibody derived from the FLAG antibody‐coupled beads used for immunoprecipitation. *, **, and *** indicate mono‐, di‐, and triubiquitinated forms of IFITM3.

3. SDS–PAGE of whole‐cell lysate input fractions from 293T cells used in (B). Immunoblotting was performed with anti‐FLAG M2, anti‐NEDD4, and anti‐actin.

4. 293T cells were transfected with 1.0 μg of pNL4‐3 and 0.5 μg of pQCXIP‐FLAG‐IFITM3 variants. Virus‐containing supernatants were harvested and 10 ng p24 equivalents were used to reinfect fresh SupT1 T cells. Productive infection was scored by immunostaining with the KC57 antibody and flow cytometry at 48 h postinfection. Mean + SD of three experiments is shown.

5. About 2.0 × 10⁵ cells were seeded and challenged with 10 ng of HIV‐1 NL4‐3 in the presence of 2 μg/ml DEAE‐dextran. Productive infection was scored by immunostaining with KC57 and flow cytometry at 48 h postinfection. The “Colobus native” sequence represents the full IFITM3 sequence identified in the colobus monkey.

6. As in (E), except that cells were challenged with influenza A virus (H1N1 PR/8/34, Charles River Laboratories), at a dose that resulted in ˜50% infection of control (Empty) cells (equivalent to 10³ TCID₅₀/0.2 ml). Infection was scored by immunostaining with an anti‐IAV NP antibody and flow cytometry at 18 h postinfection. Results are presented in a logarithmic scale. Mean + SD of 3–5 experiments is shown.

Data information: Unpaired t‐test, *P < 0.05, **P < 0.005.

Figure EV5. Further characterization of 293T 4 × 4 cells stably expressing IFITM proteins

1. 293T cells transduced to express CD4 and CXCR4 were transfected with 0.5 μg of pQCXIP‐FLAG‐IFITM3 variants, and stable expression was selected using puromycin. Corresponding mean fluorescence intensity values are shown.

2. The subcellular localization of IFITM3 variants expressed in 293T following stable transfection was assessed by confocal immunofluorescence microscopy. Representative images of two variants, human IFITM3 and human IFITM3 encoding the Δ22–24 deletion, are shown.

3. 293T cells were transfected with 0.5 μg of pQCXIP‐FLAG‐IFITM1, 2, or 3 and stable expression was selected using puromycin; 10⁵ cells were seeded and challenged with influenza A virus (H1N1 PR/8/34, Charles River Laboratories), at a dose that resulted in ˜50% infection of control (Empty) cells (equivalent to 10³ TCID₅₀/0.2 ml). Infection was scored by immunostaining with an anti‐IAV NP antibody and flow cytometry at 18 h postinfection. Mean + SD of three experiments is shown.