ABSTRACT
Ebola virus (EBOV) entry into host cells comprises stepwise and extensive interactions of the sole viral surface glycoprotein (GP) with multiple host factors. During the intricate process, following virus uptake and trafficking to late endosomal/lysosomal compartments, GP is proteolytically processed to cleaved GP (GPCL) by the endosomal proteases cathepsin B and L, unmasking GP's receptor-binding site. Engagement of GPCL with the universal filoviral intracellular receptor Niemann-Pick C1 (NPC1) eventually culminates in fusion between viral and cellular membranes, cytoplasmic escape of the viral nucleocapsid, and subsequent infection. Mechanistic delineation of the indispensable GPCL-NPC1-binding step has been severely hampered by the unavailability of a robust cell-based assay assessing interaction of GPCL with full-length endosomal NPC1. Here, we describe a novel in situ assay to monitor GPCL-NPC1 engagement in intact, infected cells. Visualization of the subcellular localization of binding complexes is based on the principle of DNA-assisted, antibody-mediated proximity ligation. Virus-receptor binding monitored by proximity ligation was contingent on GP's proteolytic cleavage and was sensitive to perturbations in the GPCL-NPC1 interface. Our assay also specifically decoupled detection of virus-receptor binding from steps post-receptor binding, such as membrane fusion and infection. Testing of multiple FDA-approved small-molecule inhibitors revealed that drug treatments inhibited virus entry and GPCL-NPC1 recognition by distinctive mechanisms. Together, here we present a newly established proximity ligation assay, which will allow us to dissect cellular and viral requirements for filovirus-receptor binding and to delineate the mechanisms of action of inhibitors on filovirus entry in a cell-based system.
