IFITM1 inhibits Henipavirus membrane fusion by trapping ephrinB2 receptors in fusion-unfavorable membrane nanodomains.

Nipah (NiV) and Hendra (HeV) viruses are prototype henipaviruses (HNV) within the family of Paramyxoviridae. They are highly pathogenic zoonotic paramyxoviruses that cause fatal encephalitis in humans, with case fatality rates of 40-70%. These viruses use the receptor-binding protein (G) to engage host receptors ephrinB2 and/or ephrinB3, while the fusion protein (F) drives membrane fusion leading to virus entry and cell-cell fusion. Interferon-inducible transmembrane (IFITM) proteins restrict the infection of many unrelated viruses at virus entry. We show that the knockdown of IFITM1 enhances HNV entry and cell-cell fusion in both human epithelial and endothelial cells, while overexpression of IFITM1 inhibits both. Using single-molecule localization microscopy, we observed that IFITM1 forms nanoclusters on the plasma membrane that are enhanced upon interferon stimulation. IFITM1 clusters spatially co-occur with GM1-enriched, raft-like membrane domains. Notably, IFITM1 interacts with ephrinB2, and redistributes ephrinB2 into larger and denser clusters, potentially within GM1-enriched regions. Using single-particle tracking, we further show that ephrinB2s lateral diffusion on the plasma membrane is confined by IFITM1, and this confinement is not reversed by amphotericin B, a membrane-permeabilizing agent that increases membrane fluidity. Our data suggest that IFITM1 sequesters ephrinB2 in fusion-unfavorable membrane domains, thereby enhancing the energy barrier for virus-cell membrane fusion and decreasing the virus-receptor encounters. SignificanceNipah (NiV) and Hendra (HeV) viruses are zoonotic paramyxoviruses that cause fatal encephalitis in humans. Here, we show that IFITM1 inhibits NiV and HeV entry at the surface of epithelial and endothelial cells. We report that IFITM1 restricts NiV entry by sequestering the host receptor ephrinB2 in membrane domains that are energetically unfavorable for virus-cell membrane fusion. IFITM1 also restricts ephrinB2 lateral diffusion on the plasma membrane, thereby reducing productive virus-receptor encounters. Notably, amphotericin B treatment, which increases membrane fluidity, did not rescue IFITM1-mediated confinement of ephrinB2 or restore NiV entry. Our data reveal a new mechanism by which IFITM1 restricts NiV infection, suggesting that its antiviral activity extends beyond simply altering membrane fluidity.