Cholesterol alters IFITM3 conformations in membrane bilayers from molecular dynamics simulations. To explore the effect of cholesterol on IFITMs in membrane bilayers, we performed molecular dynamics (MD) simulations of apo-IFITM3 and C72, C105 S-palmitoylated (palm) IFITM3 in a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membrane bilayer with or without 20% cholesterol (Fig. 1b, Supplementary Fig. 1a, Supplementary table 1). We measured tilt angles with respect to the membrane normal (i.e., Z-axis), Z positions of the center of mass of each helix with respect to the bilayer center (i.e., Z=0), as well as the interaction patterns of each residue to quantify behaviors of each helical domain namely, amphipathic helix 1 (AH1, residues 62-67), amphipathic helix 2 (AH2, residues 76-85) and transmembrane domain (TM, residues 109-131). Each helical domain shows different behaviors on addition of cholesterol in DMPC membrane systems. However, we observed similar change in trends for the helices in both apo-IFITM3 and C72, 105 S-palm IFITM3. C72, 105 S-palm IFITM3, shows more AH1 and Loop2 (residues 86-108) interactions with DMPC membrane than apo-IFITM3 (Fig. 1b, Supplementary Fig. 1a, Supplementary table 1). This is consistent with our previous studies, which showed that S-palmitoylation at Cys72 increased IFITM3 AH1 interaction with DMPC membranes23. In the presence of cholesterol, both apo and C72, 105 S-palm IFITM3 AH1 shows further increase in membrane interactions as its tilt angles are closer to 90°, a completely horizontal orientation. AH1 has a tilt angle of 86.85° for apo-IFITM3 and 77.7° for C72, 105 S-palm IFITM3 in cholesterol containing systems versus 101.53° for apo-IFITM3 and 108.4° for C72, 105 S-palm IFITM3 in DMPC-only systems (Supplementary table 2). Additionally, AH1 domain was more deeply embedded into the membrane in the presence of cholesterol, thus making more frequent interactions with lipid tails (Fig. 1c, d, Supplementary Fig. 1b). AH2, on the other hand, responses differently to the presence of cholesterol in membrane in comparison to AH1. AH2 tilt angle shows values larger than 90°, meaning that it becomes more vertical in membrane system with cholesterol (Supplementary table 2). In addition, presence of cholesterol does not affect the position of AH2 along the membrane normal in contrast to AH1 (Fig. 1d). For TM domain, the tilt angle tends to decrease in cholesterol membrane system for both apo-IFITM3 and C72, 105 S-palm IFITM3 (Fig. 1e). Such tilt angle changes are known to occur to maximize a hydrophobic match between the lipid bilayer and the transmembrane domain38. Cholesterol is known to increase the thickness and order of a lipid bilayer39, as we observed for all cholesterol containing membrane systems (Supplementary Fig. 2). Interestingly, we observe significant changes in the interaction pattern for Loop2 in membrane with and without cholesterol for both apo-IFITM3 and C72, 105 S-palm IFITM3. Loop2 is a part of the highly conserved CD225 domain which contains a basic patch consisting of R85, R87, and K88, as well as a 91GxxxG95 motif implicated in IFITM3 oligomerization and antiviral activity25,40. We see increased hydrophobic interactions in Loop2 in the DMPC system, whereas the cholesterol containing membrane system has more hydrophilic (protein-water and protein-lipid headgroup) interactions (Supplementary Fig. 3-6). Since DMPC is a fully saturated lipid and is well-mixed with rigid cholesterol, it leads to a more ordered membrane phase. This makes it difficult for Loop 2 to insert into the membrane hydrophobic core and have hydrophobic interactions. Thus, cholesterol-induced changes in IFITM3-membrane interactions might in turn influence IFITM3 interactions with other lipids and proteins as well as its activity.
To structurally characterize IFITM3 in a cholesterol-containing bilayer, we employed a bicelle membrane bilayer system. However, in the presence of full length IFITM3, the short chain lipid, 1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC) could not solubilize DMPC liposomes to form membrane bicelle. We therefore designed several truncated constructs for bicelle reconstitution. IFITM389–133, which excluded the amphipathic helices, expressed well after overnight induction, and were purified using His-affinity purification (Supplementary Fig. 7a,b). We characterized 15N-labeled IFITM389–133 in bicelles with a protein to lipid ratio of 1:50 and final q value between 0.5 and 0.6 using 2D 1H-15N TROSY spectra (Supplementary Fig. 8). We observed subtle changes in some specific residues of the transmembrane domain though full assignment of the transmembrane region residues could not be performed due to low signal. This suggests specific structural changes in the IFITM3 transmembrane domain in the presence of cholesterol in the bicelle membrane system.
Photoaffinity labeling identifies IFITM3 as a cholesterol binding immune associated protein. To investigate cholesterol binding of IFITMs in mammalian cells, we synthesized a bifunctional cholesterol analog (x-alk-chol) that has a diazirine in position 6 for crosslinking to interacting proteins on UV light exposure (Fig. 2a, Supplementary Fig. 9). This cholesterol photoaffinity probe (x-alk-chol) also has a terminal alkyne handle on the alkyl side chain for CuI-catalyzed azide-alkyne cycloaddition (CuAAC) to azide-fluorophore for fluorescence detection or azide-biotin for affinity enrichment of interacting proteins (Fig. 2a). Such photoaffinity cholesterol probes have been used to study sterol-protein interactions in cells previously41,42. Maestro modeling of x-alk-chol showed similar molecular topology as cholesterol (Supplementary Fig. 10). To probe cholesterol-binding proteins in cells, HeLa cells were treated with x-alk-chol and irradiated with UV light for x-alk-chol crosslinking with interacting proteins. The cell lysate was then subjected to CuAAC with azide-rhodamine for in-gel fluorescence profiling of x-alk-chol crosslinked proteins (Supplementary Fig. 11a). Many proteins in HeLa cells were crosslinked with x-alk-chol in a UV-dependent and dose-dependent manner (Fig. 2b and Supplementary Fig. 11b). Cells delivered excess cholesterol following x-alk-chol treatment showed lower labeling suggesting cholesterol competition with x-alk-chol (Supplementary Fig. 11c).
To enrich and identify x-alk-chol labeled proteins in HeLa cells, we subjected the cell lysate to CuAAC with azide-biotin, affinity purified with streptavidin beads and subjected to on-bead protease digestion for mass spectrometric analysis (Supplementary Fig. 12a). A summary of the proteomics data revealed enrichment of 330 proteins in UV light irradiated samples including known cholesterol binding proteins like caveolin-1 (CAV1) and Niemann-Pick disease, type C1 (NPC1), among others (Supplementary Fig. 12a)43,44. Bioinformatic analysis of the enriched proteins suggest x-alk-chol labeling of endoplasmic reticulum (ER) and plasma membrane resident membrane proteins primarily (Supplementary Fig. 12b). Many of the high confidence x-alk-chol labeled proteins, were previously identified in a proteome-labeling profile of trans-sterol probe (Supplementary Fig. 12c)41. To characterize cholesterol binding to IFITMs and other immune-associated proteins, we profiled x-alk-chol crosslinked proteins in IFN-α stimulated HeLa cells (Fig. 2c). In addition to the proteins identified in non-stimulated cells, we recovered IFITM3 and many other immune-associated proteins from the proteomic data (Fig. 2c). Western blot analysis of IFN-α stimulated and x-alk-chol treated HeLa cells showed a mobility shift for IFITM3 in UV-irradiated samples (Fig. 2d) resembling that seen with CAV1, a known cholesterol binding protein (Fig. 2d). To further confirm x-alk-chol photocrosslinking to IFITM3, the cell lysate was subjected to CuAAC with azide-biotin and affinity purified with streptavidin beads. Western blot analysis showed significant enrichment of IFITM3 and CAV1 in the UV-irradiated sample (Fig. 2e). Gene ontology analysis of the proteins enriched only in IFN-α stimulated cells identified many other membrane-associated proteins involved in lipid biosynthesis, metabolism and immunity (Supplementary Fig. 13a, b), which may warrant further investigation as many of these hits are important host factors involved in virus infection including SARS-CoV-245,46. These results show that x-alk-chol can photocrosslink many known cholesterol-binding proteins as well as capture many immune-associated proteins including IFITM3.
IFITM3-cholesterol interaction is important for antiviral activity. S-palmitoylated membrane proteins have been suggested to partition into cholesterol-rich liquid-ordered membrane microdomains47,48. IFITM3 is S-palmitoylated at Cys71, 72 and 105 (Fig. 3a). To validate IFITM3 cholesterol interaction, we performed x-alk-chol crosslinking studies with overexpressed HA tagged IFITM3 WT in HEK293T cells. In-gel fluorescence detection of anti-HA immunoprecipitated sample, shows UV-dependent x-alk-chol crosslinking of HA-IFITM3 (Fig. 3b). To identify the role of S-palmitoylation in IFITM3 cholesterol binding, we tested x-alk-chol labeling of IFITM3 PalmΔ construct, a Cys71, 72 and 105 to Ala triple mutant (Fig. 3b, c). IFITM3 PalmΔ shows significantly less x-alk-chol labeling. Bioinformatic analysis of IFITM3 orthologues from great apes and rodentia shows that IFITM3 has a putative cholesterol binding domain. IFITM3 in great apes have a cholesterol consensus domain CARC 104KCLNIWALIL113 (in pink) which lies N-terminus to the transmembrane domain (Fig. 3a). Many membrane proteins have CARC motifs or its mirror code CRAC motif to mediate interactions with cholesterol49,50. To investigate role of CARC motif in IFITM3-cholesterol interaction, we made a CARCΔ construct by replacing Lys104 with Ala and Trp109 with Ile to disrupt potential interaction with cholesterol hydroxyl group and sterol ring, respectively. We observe that x-alk-chol photocrosslinking efficiency of CARCΔ is significantly decreased (Fig. 3b, c). Thus, suggesting role of CARC motif in IFITM3-cholesterol interaction. IFITM3 Lys104 to Ala and Trp109 to Ile single mutants also show reduction in x-alk-chol photocrosslinking (Supplementary Fig. 14). IFITM3 single Cys71 and Cys72 to Ala mutants show similar x-alk-chol photocrosslinking as IFITM3 WT but Cys105 to Ala mutant shows some increase in x-alk-chol photocrosslinking. This may be due to less steric interactions at the CARC domain on mutation of Cys105 to Ala (Supplementary Fig. 15). Mouse IFITM3 does not have CARC domain conserved and shows lower levels of x-alk-chol photocrosslinking (Supplementary Fig. 16). S-palmitoylation levels of IFITM3 CARCΔ were analyzed by metabolic labeling with alk-16 labeling19. IFITM3 CARCΔ shows similar S-palmitoylation levels as IFITM3 WT (Fig. 3d, e). We also analyzed the subcellular localization of IFITM3 CARCΔ by co-expressing myc-tagged IFITM3 WT with HA-tagged IFITM3 CARCΔ. IFITM3 CARCΔ shows similar endolysosomal localization as IFITM3 WT (Fig. 3f, supplementary Fig. 17). These results suggest CARC domain of IFITM3 is crucial for its interaction with cholesterol but does not impact S-palmitoylation or subcellular localization.
To analyze the significance of IFITM3 interaction with cholesterol, we evaluated antiviral activity of IFITM3 cholesterol binding mutants against IAV and SARS-CoV-2. A549 IFITM1/2/3 KO – ACE2 cells stably expressing IFITM3 constructs were used for the infection studies (Supplementary Fig. 18). Cells expressing IFITM3 WT shows antiviral activity against IAV whereas loss of function construct IFITM3 PalmΔ has little or no activity19. Cells expressing IFITM3 CARCΔ construct shows significant loss of resistance to IAV infection (Fig. 3g, Supplementary Fig. 19). HEK293T cells expressing these IFITM3 constructs show similar trend in antiviral activity against IAV (Supplementary Fig. 20a, b). Next, we tested antiviral activity of these cell lines against a recombinant vesicular stomatitis virus bearing SARS-CoV-2 spike (rVSV-SARS-CoV-2 S)51 since recent studies show that SARS-CoV-2 spike protein interaction with cholesterol is important for virus entry and pathological syncytia formation35. However, the role of IFITMs in SARS-CoV-2 infection is unclear, since early studies show conflicting results for both inhibition of SARS-CoV-2 pseudotyped virus infection and SARS-CoV-2 spike protein mediated cell-cell fusion35,52,53,54. Furthermore, although overexpression of IFITMs restrict SARS-CoV-2 infection, endogenous levels of IFITMs was suggested to have a proviral effect55,56. Our A549 IFITM1/2/3 KO cells provides an excellent system to evaluate the activity of IFITMs in SARS-CoV-2 entry (Supplementary Fig. 18). Cells expressing IFITM3 WT shows antiviral activity against rVSV-SARS-CoV-2 S whereas IFITM3 PalmΔ or CARCΔ mutants have minimal or no antiviral activity (Fig. 3h). Thus, IFITM3-cholesterol interaction might play an important role in blocking virus fusion and release of genetic material in host cytosol during IAV and SARS-CoV-2 infection entry.
IFITMs show isoform-specific x-alk-chol photocrosslinking and antiviral activity in cells. We next analyzed cholesterol binding of human IFN-induced isoforms IFITM1, IFITM2 and IFITM3. All three IFITM isoforms have conserved Cys71, 72, 105 and cholesterol binding motif CARC (Fig. 4a). We used in-gel fluorescence profiling to evaluate photocrosslinking of IFITM isoforms with x-alk-chol. In-gel fluorescence detection and quantification normalized to protein levels showed x-alk-chol photocrosslinking to IFITM1 and IFITM3 but not with IFITM2 (Fig. 4b, c). Differential photocrosslinking IFITM2 and IFITM3 with x-alk-chol is surprising since sequence alignment of the isoforms show 83% sequence identity. These IFITM isoforms also showed similar subcellular localization by immunofluorescence confocal microscopy (Supplementary Fig. 21). However, the S-palmitoylation levels of IFITM2 and IFITM3 by alk-16 labeling correlated with x-alk-chol photocrosslinking (Fig. 4d, e). These results suggest IFITM2 and IFITM3 interaction with cholesterol maybe determined by their S-palmitoylation levels.
We then evaluated the antiviral activity of IFITM isoforms with IAV, SARS-CoV-2 and EBOV entry. IAV and SARS-CoV-2 use sialylated EGFR57 and ACE258 as entry factor respectively whereas EBOV, another virus entering from late endosomes, has been shown to use NPC1 as an entry factor59–61. For these experiments, we stably expressed the IFITM isoforms in A549 IFITM1/2/3 KO – ACE2 cells (Supplementary Fig. 22a, b). Consistent with previous reports62,63, expression of plasma membrane resident IFITM1 did not inhibit infection by these viruses that enter through low pH endosomal compartments (Fig. 4f, g, h). In contrast, we found that IFITM3 exhibits greater antiviral activity against IAV and rVSV-SARS2 S whereas IFITM2 is more active against an rVSV bearing the EBOV spike glycoprotein, GP (rVSV-EBOV GP)64 (Fig. 4f, g, h). These results suggest differential interaction with cholesterol and S-palmitoylation may impact the antiviral activity of IFITM isoforms towards different viruses.