A Virion-Based Assay for Glycoprotein Thermostability Reveals Key Determinants of Filovirus Entry and Its Inhibition

et al. 2021

mBio

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Genomic surveillance of viral isolates during the 2013–2016 Ebola virus epidemic in Western Africa, the largest and most devastating filovirus outbreak on record, revealed several novel mutations. The responsible strain, named Makona, carries an A-to-V substitution at position 82 (A82V) in the glycoprotein (GP), which is associated with enhanced infectivity in vitro. Here, we investigated the mechanistic basis for this enhancement as well as the interplay between A82V and a T-to-I substitution at residue 544 of GP, which also modulates infectivity in cell culture. We found that both 82V and 544I destabilize GP, with the residue at position 544 impacting overall stability, while 82V specifically destabilizes proteolytically cleaved GP. Both residues also promote faster kinetics of lipid mixing of the viral and host membranes in live cells, individually and in tandem, which correlates with faster times to fusion following colocalization with the viral receptor Niemann-Pick C1 (NPC1). Furthermore, GPs bearing 82V are more sensitive to proteolysis by cathepsin L (CatL), a key host factor for viral entry. Intriguingly, CatL processed 82V variant GPs to a novel product with a molecular weight of approximately 12,000 (12K), which we hypothesize corresponds to a form of GP that is pre-triggered for fusion. We thus propose a model in which 82V promotes more efficient GP processing by CatL, leading to faster viral fusion kinetics and higher levels of infectivity. IMPORTANCE The 2013–2016 outbreak of Ebola virus disease in West Africa demonstrated the potential for previously localized outbreaks to turn into regional, or even global, health emergencies. With over 28,000 cases and 11,000 confirmed deaths, this outbreak was over 50 times as large as any previously recorded. This outbreak also afforded the largest-ever collection of Ebola virus genomic sequence data, allowing new insights into viral transmission and evolution. Viral mutants arising during the outbreak have attracted attention for their potentially altered patterns of infectivity in cell culture, with potential, if unclear, implications for increased viral spread and/or virulence. Here, we report the properties of one such mutation in the viral glycoprotein, A82V, and its interplay with a previously described polymorphism at position 544. We show that mutations at both residues promote infection and fusion activation in cells but that A82V additionally leads to increased infectivity under cathepsin-limited conditions and the generation of a novel glycoprotein cleavage product.

Section: Resultsmentioning

confidence: 99%

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

Fels

Bortz

et al. 2021

mBio

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“…All FDA-approved drugs tested, amiodarone, bepridil, clomifene, sertraline, and toremifene, significantly hampered GP CL -NPC1 interaction measured by in situ PLA. Previous reports focused on bepridil, sertraline, and toremifene, which were proposed to destabilize GP’s prefusion conformation by binding into a cavity at the GP 1 -GP 2 interface in silico and in vitro ( 35 , 38 , 39 ). Our data also indicated changes in the intracellular distribution and morphology of NPC1 + LE/LY, as well as NPC1’s cholesterol transporter function following drug incubations.…”

Section: Discussionmentioning

confidence: 99%

“…The unprecedented EBOV outbreak in West Africa from 2013 to 2016 uncovered a pressing need for anti-EBOV therapeutics; since then, numerous studies screened for and characterized FDA-approved drugs with anti-filoviral activity. The most promising drug candidates, amiodarone, bepridil, clomifene, sertraline, and toremifene, were reported to block one or several steps during EBOV entry (15,(33)(34)(35)(36)(37)(38)(39). As their precise mechanisms of anti-EBOV activity still remain elusive, we examined the capacity of these inhibitors to alter intracellular GP CL -NPC1 interaction in the PLA.…”

Section: Figmentioning

confidence: 99%

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

Mittler

Jangra

et al. 2021

mBio

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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.

Section: The Gp Destabilizing Effect Of the A82v Mutation Is Unmaskedmentioning

confidence: 99%

“…Accordingly, we set out to investigate the stability of GP mutants in both uncleaved and pre-cleaved states, using an ELISA that measures the thermostability of an epitope spanning GP1 and GP2 (25) . By measuring the half-maximal melting temperature (T m ) of GPs, we found that uncleaved GPs bearing 544I were significantly less stable than their 544T counterparts ( Figure 4A and 4C ).…”

Section: The Gp Destabilizing Effect Of the A82v Mutation Is Unmaskedmentioning

confidence: 99%

A glycoprotein mutation that emerged during the 2013-2016 Ebola virus epidemic alters proteolysis and accelerates membrane fusion

Fels

Bortz

et al. 2020

Preprint

Self Cite

Genomic surveillance of viral isolates during the 2013-2016 Ebola virus epidemic in Western Africa—the largest and most devastating filovirus outbreak on record—revealed several novel mutations. The responsible strain, named Makona, carries an A to V substitution at position 82 in the glycoprotein (GP), which is associated with enhanced infectivity in vitro. Here, we investigated the mechanistic basis for this enhancement, as well as the interplay between A82V and a T to I substitution at residue 544 of GP, which also modulates infectivity in cell culture. We found that both 82V and 544I destabilize GP with the residue at 544 impacting overall stability, while 82V specifically destabilizes proteolytically cleaved GP. Both residues also promote faster kinetics of lipid mixing of the viral and host membranes in live cells, individually and in tandem, which correlates with faster times to fusion following co-localization with the viral receptor Niemann-Pick C1 (NPC1). Further, GPs bearing 82V are more sensitive to proteolysis by cathepsin L (CatL), a key host factor for viral entry. Intriguingly, CatL processed 82V variant GPs to a novel product of ~12K size, which we hypothesize corresponds to a form of GP more fully primed for fusion than previously detected. We thus propose a model in which 82V promotes more efficient GP processing by CatL, leading to faster viral fusion kinetics and higher infectivity.