S-palmitoylation and sterol interactions mediate antiviral specificity of IFITM isoforms

Abstract

Interferon-induced transmembrane proteins (IFITM1, 2 and 3) are important antiviral proteins that are active against many viruses, including influenza A virus (IAV), dengue virus (DENV), Ebola virus (EBOV), Zika virus (ZIKV) and severe acute respiratory syndrome coronavirus (SARS-CoV). IFITMs exhibit isoform-specific activity, but their distinct mechanisms of action and regulation are unclear. Since S-palmitoylation and cholesterol homeostasis are crucial for viral infections, we investigated IFITM interactions with cholesterol by molecular dynamic stimulations, nuclear magnetic resonance analysis in vitro and photoaffinity crosslinking in mammalian cells. These studies suggest that cholesterol can alter the conformation of IFITMs in membrane bilayers and directly interact with S-palmitoylated IFITMs in cells. Notably, we discovered that the S-palmitoylation levels regulate differential IFITM isoform interactions with cholesterol in mammalian cells and specificity of antiviral activity towards IAV, SARS-CoV-2 and EBOV. Our studies suggest that modulation of IFITM S-palmitoylation levels and cholesterol interaction may influence host susceptibility to different viruses.

Cholesterol binds the amphipathic helix of IFITM3 and regulates antiviral activity

“…Furthermore, they proposed that a region of IFITM3 proximal to the transmembrane domain (TMD) encodes a putative CARC consisting of 104 K CLNI W ALI L 113 (underlined residues indicate the basic, aromatic, and aliphatic residues that define a putative cholesterol binding region, as seen in certain G-protein coupled receptors (Fantini and Barrantes, 2013)). Deletion of this region led to partial loss of cholesterol analog binding, suggesting that this region of IFITM3 protein contributes to cholesterol binding in vivo (Das et al, 2021). Therefore, we tested whether a peptide overlapping with 104 K CLNI W ALI L 113 conferred potential for direct cholesterol binding in vitro .…”

“…Therefore, we suspect that engagement of cholesterol by the AH of IFITM3 similarly increases its penetrative depth and positioning within membranes and confers it with a greater capacity to stiffen and bend membranes during the virus-cell membrane fusion reaction. In fact, in the aforementioned pre-print posted during preparation of this manuscript, the authors used molecular simulations to suggest that cholesterol affects the positioning of the AH of IFITM3 in membranes (Das et al, 2021). Therefore, our description of the cholesterol binding potential of the AH likely contributes to this atomic-level observation.…”

“…However, since the AH is necessary to modify membranes in living cells (Rahman et al, 2020) and sufficient to modify artificial membranes in vitro (Guo et al, 2021), the functional consequences of cholesterol binding elsewhere in IFITM3 are unclear. Perhaps the interaction between cholesterol and 104 K CLNI W ALI L 113 affects the depth and orientation of the AH in the context of the full-length IFITM3 protein, and there is some evidence to support this possibility (Das et al, 2021). Nonetheless, 104 K CLNI W ALI L 113 is not conserved in mouse IFITM3, despite being well characterized as a restriction factor against IAV in this species (Bailey et al, 2012; Everitt et al, 2012).…”

Positive Regulation of the Antiviral Activity of Interferon-Induced Transmembrane Protein 3 by S-Palmitoylation

“…CARC domain lies on N-terminus of IFITM3, a conserved motif that mediates direct interaction between this transmembrane protein and cholesterol. Further study suggested that the CARCΔ construct of IFITM3 showed a significant loss of antiviral activity against IAV and SARS-CoV-2 infection compared with IFITM3 WT ( 87 ). In addition, cholesterol can facilitate the negative membrane curvature induced by IFITM3 resulting in increased lipid order and membrane stiffness ( 75 ).…”

“…High levels of S-palmitoylation enhanced IFITM3 interactions with cholesterol and inhibited viruses like IAV and SARS-CoV2. While with lower levels of S-palmitoylation and fewer interactions with cholesterol, IFITM2 showed more efficient anti-EBOV activity, indicating that S-palmitoylation may inhibit cholesterol-dependent viruses by regulating the interaction between IFITM3 and cholesterol ( 87 ).…”