Dataset Information

Analysis of Resistance of Ebola Virus Glycoprotein-Driven Entry Against MDL28170, An Inhibitor of Cysteine Cathepsins.

ABSTRACT: Ebola virus (EBOV) infection can cause severe and frequently fatal disease in human patients. The EBOV glycoprotein (GP) mediates viral entry into host cells. For this, GP depends on priming by the pH-dependent endolysosomal cysteine proteases cathepsin B (CatB) and, to a lesser degree, cathepsin L (CatL), at least in most cell culture systems. However, there is limited information on whether and how EBOV-GP can acquire resistance to CatB/L inhibitors. Here, we addressed this question using replication-competent vesicular stomatitis virus bearing EBOV-GP. Five passages of this virus in the presence of the CatB/CatL inhibitor MDL28170 were sufficient to select resistant viral variants and sequencing revealed that all GP sequences contained a V37A mutation, which, in the context of native GP, is located in the base of the GP surface unit. In addition, some GP sequences harbored mutation S195R in the receptor-binding domain. Finally, mutational analysis demonstrated that V37A but not S195R conferred resistance against MDL28170 and other CatB/CatL inhibitors. Collectively, a single amino acid substitution in GP is sufficient to confer resistance against CatB/CatL inhibitors, suggesting that usage of CatB/CatL inhibitors for antiviral therapy may rapidly select for resistant viral variants.

SUBMITTER: Hoffmann M

PROVIDER: S-EPMC6963435 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Analysis of Resistance of Ebola Virus Glycoprotein-Driven Entry Against MDL28170, An Inhibitor of Cysteine Cathepsins.

Hoffmann Markus M Kaufmann Svenja Victoria SV Fischer Carina C Maurer Wiebke W Moldenhauer Anna-Sophie AS Pöhlmann Stefan S

Pathogens (Basel, Switzerland) 20191015 4

Ebola virus (EBOV) infection can cause severe and frequently fatal disease in human patients. The EBOV glycoprotein (GP) mediates viral entry into host cells. For this, GP depends on priming by the pH-dependent endolysosomal cysteine proteases cathepsin B (CatB) and, to a lesser degree, cathepsin L (CatL), at least in most cell culture systems. However, there is limited information on whether and how EBOV-GP can acquire resistance to CatB/L inhibitors. Here, we addressed this question using repl ...[more]

PMID: 31618932

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Project description:The recent Ebola virus (EBOV) epidemic in West Africa demonstrates the potential for a significant public health burden caused by filoviral infections. No vaccine or antiviral is currently FDA approved. To expand the vaccine options potentially available, we assessed protection conferred by an EBOV vaccine composed of vesicular stomatitis virus pseudovirions that lack native G glycoprotein (VSV?G) and bear EBOV glycoprotein (GP). These pseudovirions mediate a single round of infection. Both single-dose and prime/boost vaccination regimens protected mice against lethal challenge with mouse-adapted Ebola virus (ma-EBOV) in a dose-dependent manner. The prime/boost regimen provided significantly better protection than a single dose. As N-linked glycans are thought to shield conserved regions of the EBOV GP receptor-binding domain (RBD), thereby blocking epitopes within the RBD, we also tested whether VSV?G bearing EBOV GPs that lack GP1 N-linked glycans provided effective immunity against challenge with ma-EBOV or a more distantly related virus, Sudan virus. Using a prime/boost strategy, high doses of GP/VSV?G partially or fully denuded of N-linked glycans on GP1 protected mice against ma-EBOV challenge, but these mutants were no more effective than wild-type (WT) GP/VSV?G and did not provide cross protection against Sudan virus. As reported for other EBOV vaccine platforms, the protection conferred correlated with the quantity of EBOV GP-specific Ig produced but not with the production of neutralizing antibodies. Our results show that EBOV GP/VSV?G pseudovirions serve as a successful vaccination platform in a rodent model of Ebola virus disease and that GP1 N-glycan loss does not influence immunogenicity or vaccination success.IMPORTANCE The West African Ebola virus epidemic was the largest to date, with more than 28,000 people infected. No FDA-approved vaccines are yet available, but in a trial vaccination strategy in West Africa, recombinant, infectious VSV encoding the Ebola virus glycoprotein effectively prevented virus-associated disease. VSV?G pseudovirion vaccines may prove as efficacious and have better safety, but they have not been tested to date. Thus, we tested the efficacy of VSV?G pseudovirions bearing Ebola virus glycoprotein as a vaccine platform. We found that wild-type Ebola virus glycoprotein, in the context of this platform, provides robust protection of EBOV-challenged mice. Further, we found that removal of the heavy glycan shield surrounding conserved regions of the glycoprotein does not enhance vaccine efficacy.

Project description: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.

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Dataset Information

Analysis of Resistance of Ebola Virus Glycoprotein-Driven Entry Against MDL28170, An Inhibitor of Cysteine Cathepsins.

Publications

Analysis of Resistance of Ebola Virus Glycoprotein-Driven Entry Against MDL28170, An Inhibitor of Cysteine Cathepsins.

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