ABSTRACT
Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the prefusion conformers of class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.IMPORTANCE The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.
Subject(s)
Ebolavirus/drug effects , Niemann-Pick C1 Protein/antagonists & inhibitors , Receptors, Virus/antagonists & inhibitors , Viral Envelope Proteins/antagonists & inhibitors , Viral Fusion Proteins/antagonists & inhibitors , Virion/drug effects , Animals , Binding Sites , Biological Assay , Chlorocebus aethiops , Clomiphene/chemistry , Clomiphene/pharmacology , Ebolavirus/chemistry , Ebolavirus/genetics , Ebolavirus/metabolism , Epitopes/chemistry , Epitopes/genetics , Epitopes/metabolism , Hot Temperature , Hydrogen-Ion Concentration , Molecular Docking Simulation , Niemann-Pick C1 Protein/chemistry , Niemann-Pick C1 Protein/genetics , Niemann-Pick C1 Protein/metabolism , Protein Binding/drug effects , Protein Interaction Domains and Motifs , Protein Stability , Protein Structure, Tertiary , Receptors, Virus/chemistry , Receptors, Virus/genetics , Receptors, Virus/metabolism , Tamoxifen/analogs & derivatives , Tamoxifen/chemistry , Tamoxifen/pharmacology , Toremifene/chemistry , Toremifene/pharmacology , Vero Cells , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/genetics , Viral Fusion Proteins/metabolism , Virion/chemistry , Virion/genetics , Virion/metabolismABSTRACT
In the version of this Article originally published, the bat species for 12 individuals were incorrectly identified in Supplementary Table 1 and 2. After resequencing the MT-CytB and MT-CO1 segments and reviewing the data, the authors have corrected the errors for these 12 animals. In the amended version of the Supplementary Information, Supplementary Tables 1 and 2 have been replaced to include the corrected host species information. None of the 12 bats affected were positive for the Bombali virus, and the conclusions of the study are therefore unchanged.
ABSTRACT
Here we describe the complete genome of a new ebolavirus, Bombali virus (BOMV) detected in free-tailed bats in Sierra Leone (little free-tailed (Chaerephon pumilus) and Angolan free-tailed (Mops condylurus)). The bats were found roosting inside houses, indicating the potential for human transmission. We show that the viral glycoprotein can mediate entry into human cells. However, further studies are required to investigate whether exposure has actually occurred or if BOMV is pathogenic in humans.
