Direct Intracellular Visualization of Ebola Virus-Receptor Interaction by In Situ Proximity Ligation

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. IMPORTANCE Ebola virus causes episodic but increasingly frequent outbreaks of severe disease in Middle Africa, as shown by the recently overcome second largest outbreak on record in the Democratic Republic of Congo. Despite considerable effort, FDA-approved anti-filoviral therapeutics or targeted interventions are not available yet. Virus host-cell invasion represents an attractive target for antivirals; however, our understanding of the inhibitory mechanisms of novel therapeutics is often hampered by fragmented knowledge of the filovirus-host molecular interactions required for viral infection. To help close this critical knowledge gap, here, we report an in situ assay to monitor binding of the EBOV glycoprotein to its receptor NPC1 in intact, infected cells. We demonstrate that our in situ assay based on proximity ligation represents a powerful tool to delineate receptor-viral glycoprotein interactions. Similar assays can be utilized to examine receptor interactions of diverse viral surface proteins whose studies have been hampered until now by the lack of robust in situ assays.

A Glycoprotein Mutation That Emerged during the 2013–2016 Ebola Virus Epidemic Alters Proteolysis and Accelerates Membrane Fusion

“…One of the most striking and widely reported phenotypes associated with the A82V mutation is increased resistance to the small-molecule viral inhibitor 3.47, which targets the GP-NPC1 interaction ( 12 , 22 , 23 ). We observed that the polymorphisms at positions 82 and 544 made independent and additive contributions to viral sensitivity to 3.47.…”

“…Because CatL can support EBOV GP-dependent entry ( 13 , 14 , 29 ), we next sought to investigate the effect of the position 82/544 polymorphisms on viral entry under CatL-limited conditions. To overcome the challenges associated with selective inhibition of CatL in cells with irreversible activity-based inhibitors, we previously generated U2OS CatL knockout (KO) cells through CRISPR/Cas9 genome engineering ( 23 ). We then tested the kinetics of lipid mixing of the GP Mayinga variants that displayed the greatest difference on wild-type (WT) U2OS cells.…”

Multiple cell types support productive infection and dynamic translocation of infectious Ebola virus to the surface of human skin

“…To evaluate if these explants serve as robust multicellular intermediate model systems that offer insight into small molecule efficacy in a readily accessible human tissue, we assessed several well-established EBOV inhibitors for their ability to block EBOV or rVSV/EBOV GP infection in skin explants. The cysteine protease inhibitor E64, the endosomal Ca2 + channel blocking agent tetrandrine, and the pro-inflammatory cytokine interferon-γ (IFN-γ) were evaluated as they have been shown previously to block infection (16,20,45). Skin explants were submerged in media containing the appropriate concentration of inhibitor overnight at 37°C to allow tissue penetration.…”

Viral Membrane Fusion: A Dance Between Proteins and Lipids

“…Simple receptor-triggered fusion events occur at neutral pH leading to virus entry through the plasma membrane. Low pH-requiring class I fusion proteins from the arenavirus ( 10 , 42 , 43 ), filovirus ( 44 – 46 ) and arterivirus ( 47 ) families engage endosomal receptors. For Lassa fever virus (LASV), the endosomal receptor, Lamp1, is not absolutely required but rather upwardly shifts the fusion pH threshold (from ~5.0 to ~5.5), thereby enhancing infection ( 48 ).…”