Growth-Adaptive Mutations in the Ebola Virus Makona Glycoprotein Alter Different Steps in the Virus Entry Pathway

Abstract

The (EBOV) glycoprotein (GP) is cleaved into two subunits (GP1 and GP2) that are both required for virus attachment and entry into cells. Sequence changes in the GP have been proposed to increase pathogenesis and to alter virus growth properties. Mutations in GP acquired during EBOV tissue culture passage have also been reported to change virus growth properties. Here, we report the isolation of six amino acid mutations in EBOV GP that spontaneously appeared during recovery and passage of an EBOV-Makona GP-pseudotyped vesicular stomatitis virus (VSV), two of which also occur during passage of EBOV clinical isolates in tissue culture. Each of the six mutations resulted in increased virus growth in monkey and human cell lines. All mutations are located in the GP2 fusion subunit and increase entry kinetics of EBOV virus-like particles (VLPs). The gain-of-entry function mapped to two mechanistic phenotypes. Mutations in heptad repeat 1 (HR1) decreased the requirement for cathepsin B activity for viral infection. Mutations directly within the fusion loop increased entry kinetics without altering the cathepsin B dependence. Several mutations in the fusion loop were substitutions of residues present in other ebolavirus glycoproteins, illustrating the evolutionary paths for maintaining an optimally functioning fusion loop under selection pressure. (EBOV) is the causative agent of the highly lethal Ebola virus disease and poses a significant threat to the global health community. Approved antivirals against EBOV are lacking; however, promising therapies targeting the EBOV glycoprotein are being developed. Efficacy testing of these candidate therapeutics relies on EBOV laboratory stocks, which when grown in tissue culture may acquire mutations in the glycoprotein. These mutations can produce inaccurate results in therapeutic testing. Until recently, distinguishing between tissue culture mutations and naturally occurring polymorphisms in EBOV GP was difficult in the absence of consensus clinical GP sequences. Here, we utilize recombinant VSV (rVSV) pseudotyped with the consensus clinical EBOV Makona GP to identify several mutations that have emerged or have potential to emerge in EBOV GP during tissue culture passage. Identifying these mutations informs the EBOV research community as to which mutations may arise during preparation of laboratory virus stocks.

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

“…In order to validate these pseudotyped viruses, we first measured their infectivity in Vero cells. The A82V and T544I mutations, both alone and together, promoted higher levels of infectivity ( Figure 1A ), as previously reported (15)(16)(17)(18) . The differences in infectivity did not stem from differences in GP density on the viral surface, as all four variants demonstrated comparable levels of GP incorporation into particles ( Figure 1B ).…”

“…Previous findings indicate that both the A82V and T544I mutations influence the CatB dependence of viral entry. While T544I alone does not promote resistance to CA074, the residue at position 544 does influence the overall dependence on CatB (10,18) . Here, we found that only the 82V/544I variant was resistant to the CatB inhibitor CA074 ( Figure 6A ), largely consistent with Wang and co-workers' observations.…”

“…We propose that polymorphisms at position 544 primarily influence global GP stability, which may have ramifications for initial proteolytic cleavages in GP or even for fusogenic rearrangement. Due to its location in the GP2 internal fusion loop, residue 544 has been speculated to alter fusogenicity and/or bilayer insertion (10,17,18,36) . In GP peptides at low pH, 544I forms part of a "hydrophobic fist" together with 529L…”

“…Another polymorphism in EBOV GP, at position 544, has also been shown to influence viral entry in tissue culture, with 544I conferring enhanced infectivity relative to 544T (15)(16)(17)(18) .…”

An exploration of conditions proposed to trigger the Ebola virus glycoprotein for fusion

“…In conclusion, our study highlights the complexity of the EBOV fusion mechanism and adds support to the proposal that additional cues are necessary (after cleavage of GP to GPcl, binding to NPC1 and exposure to low pH) for EBOV to open, expand and sustain a robust fusion pore. Future studies are aimed at identifying the putative missing factor(s) and testing other filoviral GPs that may be more prone to fusion, as suggested in a recent report by Ruedas et al [61]. Fig 1A. The surfaces of effector and target HEK293T/17 cells were biotinylated and analyzed for surface exposed EBOV-GP1 (A) or NPC1-C-loop (B) as described in the Methods section, blotting for, respectively.…”