Jolley, A. A. | Search bioRxiv

bioRxiv2024

SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

The CD225/Dispanins superfamily consists of membrane proteins that regulate vesicular transport and membrane fusion events driving neurotransmission, glucose transport, and antiviral immunity. However, how the CD225 domain controls membrane trafficking was unknown. We reveal that the CD225 domain contains a SNARE-like motif that enables interaction with cellular SNARE fusogens. Proline rich transmembrane protein 2 (PRRT2) encodes a SNARE-like motif that enables interaction with neuronal SNARE proteins, and mutations therein disrupt SNARE binding and are linked to neurological disease. Another CD225 member, interferon-induced transmembrane protein 3 (IFITM3), protects cells against Influenza A virus infection. IFITM3 interacts with SNARE proteins that mediate late endosome-late endosome (homotypic) fusion and late endosome-lysosome (heterotypic) fusion. IFITM3 binds to syntaxin 7 (STX7) in cells and in vitro, and mutations that abrogate STX7 binding cause loss of antiviral activity against Influenza A virus. Mechanistically, IFITM3 disrupts assembly of the SNARE complex controlling homotypic fusion and accelerates the trafficking of endosomal cargo to lysosomes. Our results suggest that SNARE modulation plays a previously unrecognized role in the diverse functions performed by CD225 proteins.

cell biology↗

bioRxiv2022

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 inhibition 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 antiviral 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 contribute 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.

immunology↗