IFITM Proteins Restrict Viral Membrane Hemifusion

Abstract

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection.

TIM-family proteins inhibit HIV-1 release

“…The experiments were performed as described (53,54). Briefly, 293T cells were transfected with plasmids encoding HIV-1 proviral DNAs or HIV-1 Gag, along with plasmids expressing TIMs or PS receptors.…”

The Laurdan Spectral Phasor Method to Explore Membrane Micro-heterogeneity and Lipid Domains in Live Cells

“…A more rigid membrane environment influences the lateral diffusion and concentration of membrane proteins [4], which in turn regulates interactions among proteins and therefore the efficiency of signaling pathways through the formation of membrane domains. Some examples are viral entry/ budding [5], cell polarization and adhesion [6], membrane trafficking [7,8], and B cell signaling [9]. The membrane lipid composition defines the degree of lipid packing, the thickness of the bilayer, and the rotational freedom that the lipids have in the bilayer.…”

Les IFITM, un obstacle commun à de nombreux virus

“…Pour l'instant, ces mécanismes ont été décrits pour IFITM3, mais comme les séquences et les localisations intracellulaires des IFITM2 et -3 sont similaires, il est probable qu'ils s'appliquent aussi à IFITM2. En revanche, IFITM1 possède un domaine [34]. L'étape de fusion se décompose elle-même en plusieurs processus (Figure 3).…”

“…Deux études ont cherché à déterminer quelle était l'étape bloquée par les IFITM. La première suggère que le processus d'hémifusion n'a pas lieu ; elle se fonde sur le fait qu'une drogue (chlorpromazine, Cpz), qui favorise le passage de l'état d'hémifusion à l'ouverture du pore, ne lève pas la restriction imposée par IFITM3 [34]. La seconde suggère que l'ouverture du pore est inhibée ; elle utilise des fluorophores différents pour la membrane virale et l'intérieur du virion, et observe la fusion des membranes cellulaire et virale, mais l'absence de libération du contenu des virus dans le cytoplasme cellulaire [35].…”

“…D'une part, les IFITM participent à la rigidification des membranes et, en favorisant une courbure positive, conduiraient à une diminution du processus de fusion [34]. Une seconde équipe a mis en évidence une interaction entre VAPA (vesicle-associated membrane protein-associated protein A) et IFITM3, inhibant l'interaction VAPA-OSBP (oxysterolbinding protein) qui est primordiale pour l'homéosta-sie intracellulaire du cholestérol [30].…”