IFITM1-mediated restriction of henipavirus entry is associated with ephrinB2 reorganization and confinement in 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 to 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 enlarged upon interferon stimulation. IFITM1 clusters spatially co-occur with GM1-enriched, raft-like membrane domains. EphrinB2, a lipid raft resident, organizes into larger and denser clusters when associated with IFITM1, suggesting a preferential partitioning into membrane domains organized and/or influenced by IFITM1. Using single-particle tracking, we further show that lateral diffusion of ephrinB2 on the plasma membrane is confined in the presence of IFITM1. This confinement is not reversed by amphotericin B, nor is IFITM1-mediated NiV restriction. Our data show spatial reorganization and confinement of ephrinB2 within membrane domains that may be organized and/or influenced by IFITM1. These changes may contribute to HNV restriction, potentially by increasing the energy barrier for virus-cell membrane fusion and reducing productive virus-receptor encounters.