Direct intracellular visualization of Ebola virus-receptor interaction byin situproximity 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 GPCLby the endosomal proteases cathepsin B and L unmasking GP’s receptor-binding site. Engagement of GPCLwith 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 GPCLwith full-length endosomal NPC1.Here, we describe a novelin situassay 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 a currently ongoing outbreak 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 anin situassay to monitor binding of the EBOV glycoprotein to its receptor NPC1 in intact, infected cells. We demonstrate that ourin situassay 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 robustin situassays..

Medienart:

Preprint

Erscheinungsjahr:

2021

Erschienen:

2021

Enthalten in:

bioRxiv.org - (2021) vom: 15. Dez. Zur Gesamtaufnahme - year:2021

Sprache:

Englisch

Beteiligte Personen:

Mittler, Eva [VerfasserIn]
Alkutkar, Tanwee P [VerfasserIn]
Jangra, Rohit K [VerfasserIn]
Chandran, Kartik [VerfasserIn]

Links:

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doi:

10.1101/2020.05.05.080085

funding:

Förderinstitution / Projekttitel:

PPN (Katalog-ID):

XBI017770041