Cholesterol Binds the Amphipathic Helix of IFITM3 and Regulates Antiviral Activity

The interferon-induced transmembrane (IFITM) proteins broadly inhibit the entry of diverse pathogenic viruses, including Influenza A virus (IAV), Zika virus, HIV-1, and SARS coronaviruses by inhibiting virus-cell membrane fusion. IFITM3 was previously shown to disrupt cholesterol trafficking, but the functional relationship between IFITM3 and cholesterol remains unclear. We previously showed that inhi-bition of IAV entry by IFITM3 is associated with its ability to promote cellular membrane rigidity, and these activities are functionally linked by a shared requirement for the amphipathic helix (AH) found in the intramembrane domain (IMD) of IFITM3. Furthermore, it has been shown that the AH of IFITM3 alters lipid membranes in vitro in a cholesterol-dependent manner. Therefore, we aimed to elucidate the relationship between IFITM3 and cholesterol in more detail. Using a fluorescence-based in vitro binding assay, we found that a peptide derived from the AH of IFITM3 directly interacted with the cholesterol analog, NBD-cholesterol, while other regions of the IFITM3 IMD did not, and native cholesterol competed with this interaction. In addition, recombinant full-length IFITM3 protein also exhibited NBD-cholesterol binding activity. Importantly, previously characterized mutations within the AH of IFITM3 that strongly inhibit antivi-ral function (F63Q and F67Q) disrupted AH structure in solution, inhibited cholesterol binding in vitro, and restricted bilayer insertion in silico. Our data suggest that direct interactions with cholesterol may con-tribute to the inhibition of membrane fusion pore formation by IFITM3. These findings may facilitate the design of therapeutic peptides for use in broad-spectrum antiviral therapy. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Automated summary

The study reveals that the interferon-induced transmembrane protein 3 (IFITM3) directly interacts with cholesterol, which may play a crucial role in inhibiting virus-cell membrane fusion. This finding could facilitate the design of therapeutic peptides for broad-spectrum antiviral therapy.