A multiplex interactome of Ebola virus proteins reveals TM9SF2 as a cell-surface attachment factor that promotes viral entry.

The high lethality of the Ebola virus (EBOV) underscores the critical need to understand its molecular lifecycle. Here, we systematically mapped EBOV-host interactions using affinity purification-mass spectrometry (AP-MS) and proximity labeling, generating a comprehensive protein-protein interaction (PPI) network, of which 1728 are high-confidence core PPIs involving nine EBOV proteins and 914 human interactors. Notably, we identified transmembrane 9 superfamily member 2 (TM9SF2) as a novel attachment factor that facilitates EBOV glycoprotein (GP)-mediated entry. Mechanistic studies demonstrated that the receptor-binding domain (RBD) of EBOV GP associates with the extracellular domain (ECD) of TM9SF2, enhancing cellular adhesion and viral attachment. Moreover, the anti-TM9SF2 antibodies effectively attenuated EBOV GP-mediated pseudovirus entry and infection in vitro. Additionally, Tm9sf2 +/ - knockout mice exhibited significantly reduced susceptibility to EBOV pseudoviruses. These findings provide critical insights into EBOV-host interactions and establish TM9SF2 as a promising therapeutic target for antiviral intervention.