A virion-based assay for glycoprotein thermostability reveals key determinants of filovirus entry and its inhibition

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 pre-fusion 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 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 pre-fusion 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 CL . 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. ImportanceThough a vaccine for Ebola virus has been approved by the FDA within the past year, no FDA-approved therapeutics are available to treat infections by Ebola virus or other filoviruses. The development of such countermeasures is challenged by our limited understanding of the mechanism by which Ebola virus enters cells, especially at the final step of membrane fusion. The sole surface-exposed viral protein, GP, mediates key steps in virus entry, including 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 structural 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 forms of GP with enhanced stability that cou...

Two Distinct Lysosomal Targeting Strategies Afford Trojan Horse Antibodies With Pan-Filovirus Activity

“…Elgon-Musoke) were previously described ( 2 , 5 , 27 , 34 – 37 ). An rVSV encoding eGFP in the first position and MLAV GP (MLAV/Rousettus-wt/CHN/2015/Sharen-Bat9447-1; GenBank Accession no: KX371887) was cloned and rescued as above ( 2 , 5 , 27 , 38 – 41 ).…”