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.