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

ABSTRACT: The Zaire ebolavirus (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.IMPORTANCEZaire ebolavirus (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.

SUBMITTER: Ruedas JB

PROVIDER: S-EPMC6146795 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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

Ruedas John B JB Arnold Catherine E CE Palacios Gustavo G Connor John H JH

Journal of virology 20180912 19

The <i>Zaire ebolavirus</i> (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 recove ...[more]

PMID: 30021890

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Project description:Ebola virus (EBOV) causes highly lethal disease outbreaks against which no FDA-approved countermeasures are available. Although many host factors exploited by EBOV for cell entry have been identified, including host cell surface phosphatidylserine receptors, endosomal cysteine proteases, and the lysosomal cholesterol trafficking protein NPC1, key questions remain. Specifically, late entry steps culminating in viral membrane fusion remain enigmatic. Here, we investigated a set of glycoprotein (GP) mutants previously hypothesized to be entry defective and identified one mutation, R64A, that abolished infection with no apparent impact on GP expression, folding, or viral incorporation. R64A profoundly thermostabilized EBOV GP and rendered it highly resistant to proteolysis in vitro Forward-genetics and cell entry studies strongly suggested that R64A's effects on GP thermostability and proteolysis arrest viral entry at least at two distinct steps: the first upstream of NPC1 binding and the second at a late entry step downstream of fusion activation. Concordantly, toremifene, a small-molecule entry inhibitor previously shown to bind and destabilize GP, may selectively enhance the infectivity of viral particles bearing GP(R64A) at subinhibitory concentrations. R64A provides a valuable tool to further define the interplay between GP stability, proteolysis, and viral membrane fusion; to explore the rational design of stability-modulating antivirals; and to spur the development of next-generation Ebola virus vaccines with improved stability.IMPORTANCE Ebola virus is a medically relevant virus responsible for outbreaks of severe disease in western and central Africa, with mortality rates reaching as high as 90%. Despite considerable effort, there are currently no FDA-approved therapeutics or targeted interventions available, highlighting the need of development in this area. Host-cell invasion represents an attractive target for antivirals, and several drug candidates have been identified; however, our limited understanding of the complex viral entry process challenges the development of such entry-targeting drugs. Here, we report on a glycoprotein mutation that abrogates viral entry and provides insights into the final steps of this process. In addition, the hyperstabilized phenotype of this mutant makes it useful as a tool in the discovery and design of stability-modulating antivirals and next-generation vaccines against Ebola virus.

Dataset Information

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

Publications

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

Similar Datasets

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