Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to its oligomeric state

Abstract

The interferon-inducible transmembrane (IFITM) proteins belong to the Dispanin/CD225 family and inhibit diverse virus infections. IFITM3 reduces membrane fusion between cells and virions through a poorly characterized mechanism. Mutation of proline rich transmembrane protein 2 (PRRT2), a regulator of neurotransmitter release, at glycine-305 was previously linked to paroxysmal neurological disorders in humans. Here, we show that glycine-305 and the homologous site in IFITM3, glycine-95, drive protein oligomerization from within a GxxxG motif. Mutation of glycine-95 (and to a lesser extent, glycine-91) disrupted IFITM3 oligomerization and reduced its antiviral activity against Influenza A virus. An oligomerization-defective variant was used to reveal that IFITM3 promotes membrane rigidity in a glycine-95-dependent and amphipathic helix-dependent manner. Furthermore, a compound which counteracts virus inhibition by IFITM3, amphotericin B, prevented the IFITM3-mediated rigidification of membranes. Overall, these data suggest that IFITM3 oligomers inhibit virus-cell fusion by promoting membrane rigidity.

Membrane Interference Against HIV-1 by Intrinsic Antiviral Factors: The Case of IFITMs

“…Lassa virus has been described to use late rather than early endosomes to access the cell cytoplasm [ 53 , 54 ] and a predominant distribution of IFITMs in the latter could potentially explain the absence of IFITMs in Lassa virus-containing vesicles. However, IFITM2/3 appear equally distributed between late and early endosomes [ 55 ]. As such, these results raise a fundamental question that remains to be addressed: are endosomes more heterogeneous than expected and IFITMs are specific markers of a yet uncharacterized endosomal subpopulation, or else are IFITMs driving specific changes in endosomes in which they are embedded that endows them with specific features?…”

“…Membranes of cells expressing IFITM proteins are more rigid and this has been determined upon Laurdan staining coupled with two-photon and fluorescence-lifetime imaging microscopy (Laurdan is a hydrophobic fluorescent probe sensitive to lipid phases), as well as after use of a novel fluorescent lipid tension FliptR reporter [ 36 , 37 , 55 , 60 , 61 ]. In agreement with a model in which IFITMs induce more rigid membranes, amphotericin B, an antifungal antibiotic that instead fluidifies them, has been shown to oppose the effects of IFITM3 against different viruses [ 38 , 55 , 62 ].…”

“…At present, several lines of evidence argue in favor of a direct physical action of IFITMs on membrane rigidity: (i) the restriction of incoming viruses requires the colocalization with IFITM-decorated endosomes [ 38 , 39 , 44 ]; (ii) IFITM oligomerization is required for membrane rigidification and this correlates with viral restriction [ 55 , 64 ]; (iii) IFITM3 induces negative curvature in vitro which is consistent with membrane rigidification [ 60 ].…”

“…Paradoxically, the same experiments on the ordered phase of lipids mentioned above lend ground to the hypothesis of an indirect effect of IFITMs on the alterations of biophysical properties of membranes. Indeed, partly due to technical challenges in measuring Laurdan changes in endosomal membranes, increased lipid order has been essentially measured at the plasma membrane [ 36 , 55 ]. According to our data, the amount of IFITM2/3 at the plasma membrane in different cell types is at least ten-fold lower than it is at internal membranes [ 34 ], suggesting either that very low levels of IFITM3 are sufficient to durably change the biophysical properties of membranes, or else that the lipid phase ordering effects measured at the plasma membrane are the additive result of both a direct and an indirect effect of IFITMs.…”

IFITM3 incorporation sensitizes influenza A virus to antibody-mediated neutralization

“…The molecular mechanisms of IFITM-mediated viral inhibition are still under investigation. Current literature agree that cellular membranes show increased positive curvature and decreased membrane fluidity upon incorporation of IFITMs, offering a convincing explanation as to why IFITMs block fusion between cellular and viral membranes (Li et al, 2013;Lin et al, 2013;Rahman et al, 2020;Yount et al, 2012). In addition to their biophysical properties, IFITMs also rely on appropriate subcellular localization to exert their antiviral function.…”