Activation of Toll-like receptor 4 by Ebola virus-shed glycoprotein is direct and requires the internal fusion loop but not glycosylation

Infection by the Ebola virus, a member of the Filoviridae family of RNA viruses, leads to acute viral hemorrhagic fever. End-stage Ebola virus disease is characterized by a cytokine storm that causes tissue damage, vascular disintegration, and multi-organ failure. Previous studies showed that a shed form of the viral spike glycoprotein (sGP1,2) drives this hyperinflammatory response by activating Toll-like receptor 4 (TLR4). Here, we find that glycosylation is not required for activation of TLR4 by sGP1,2 and identify the internal fusion loop (IFL) as essential for inflammatory signaling. sGP1,2 competes with lipid antagonists of TLR4, and the IFL interacts directly with TLR4 and co-receptor MD2. Together, these findings indicate that sGP1,2 activates TLR4 analogously to bacterial agonist lipopolysaccharide (LPS) by binding into a hydrophobic pocket in MD2 and promoting the formation of an active heterotetramer. This conclusion is supported by docking studies that predict binding sites for sGP1,2 on TLR4 and MD2.