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

Abstract

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.

TIM-1 Augments Cellular Entry of Ebola Virus Species and Mutants, Which Is Blocked by Recombinant TIM-1 Protein

“…Recently, several mutated Ebola virus species have been found to evade antibody attack ( 5 – 11 ). EBOV harboring GP-K510E and GP-D552N was refractory to ADI-15946 and MIL77-2 treatment, respectively ( 30 , 36 ).…”

“…EBOV strains harboring GP mutations (A82V and T544I) have been shown to reduce the stability of the prefusion conformation of GP compared to their parental strain and thus enhance infection by decreasing the threshold for activation of membrane fusion activity triggered by host factors cathepsin B and Niemann-Pick C1 ( 6 , 7 , 53 ). Also, some researchers attributed the enhanced infection to I544 stabilizing the primed GP structure by increasing the hydrophobicity around residue 544, which is critical for virus-host membrane fusion ( 9 , 54 , 55 ).…”

“…Ebola virus glycoprotein (GP) is a surface viral protein and is responsible for viral receptor binding and cell entry. Recent studies have shown that EBOV strains harboring two GP mutations—alanine to valine at position 82 (A82V) and threonine to isoleucine at position 544 (T544I)—may cause an increase in viral infectivity in humans ( 5 – 11 ).…”

Probe-Free Identification of RNA Virus Variants with Point Mutations by Surface-Enhanced Raman Spectroscopy

“…Envelope proteins are involved in the entry into target host cells, generally affecting the infectivity and possibly the pathogenicity of RNA viruses . Newly emerging RNA viruses would also likely have point mutations in their envelope proteins . To prepare viral particles displaying mutated envelope proteins for SERS analysis, we introduced point mutations into the genes encoding the WSN strain HA and NA by error-prone PCR using the genes as templates.…”

The Key Role of Lysosomal Protease Cathepsins in Viral Infections

“…Cathepsins B and L have been implicated in the proteolytic cleavage of the viral glycoprotein (GP) of the Ebola virus (EBOV) that facilitates virus interaction with the cellular receptor(s) and its entry into target cells [ 63 , 64 , 65 ]. Interestingly, faster viral fusion kinetics and enhanced infectivity of the Ebola strain named Makona, which carries an A-to-V substitution at position 82 (A82V) in the GP, have been correlated with a more efficient GP processing by cathepsin L [ 66 ]. Both cathepsins B and L seem to be also involved in the entry initial step of infection by human papillomavirus type 16 virus (HPV16) [ 67 , 68 , 69 ].…”