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. envelope with the limiting membrane of endolysosomes [12], allowing delivery of the viral genome into the host cell cytoplasm. Binding and membrane fusion are carried out by different subunits of GP. The N-terminal surface unit, GP1, binds to host cell receptors via a receptor-binding domain (RBD), while the C-terminal transmembrane unit, GP2, harbors the functional elements required for membrane fusion, including a fusion peptide and heptad repeats [10]. Although GP1 can bind to attachment factors on the cell surface [13,14], only the interaction with the intracellular receptor Niemann-Pick C1 (NPC1) within host cell endolysosomes is essential for EBOV entry [13][14][15][16]. This interaction critically depends on previous processing of EBOV-GP by the endolysosomal, pH-dependent cysteine proteases cathepsin B (CatB) and cathepsin L (CatL) [15][16][17][18]. Processing removes a glycan cap and a mucin-like domain (MLD) from GP1 and results in a primed form of GP. Primed GP still contains the RBD linked to GP2 via a disulfide bond [19,20] and can fuse the viral membrane with an endolysosomal membrane upon receipt of a trigger. However, the nature of this trigger remains to be identified [21][22][23][24].In the light of the important role of CatB/CatL in EBOV-GP-driven entry, inhibitors targeting these enzymes might hold therapeutic potential, although it should be stated that CatB/CatL dependence varies between filoviruses [25,26] and target cell types [27] and was not observed in experimentally infected mice [28]. A previous study employed a replication-competent VSV chimera bearing EBOV-GP without MLD (VSV-EBOV) to examine resistance development against the CatB inhibitor CA074 [29]. Resistance development was observed after two pa...

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection

“…We observed dose-dependent inhibition of SARS-CoV-2 induced cell death and virus replication with Calpain Inhibitor III, which targets Cathepsin L ( Fig. 5A , E – F ) ( 43 , 44 ). Surprisingly, we observed strong cell toxicity of the PIKFyve kinase inhibitor APY0201 even at 0.6 μM concentration.…”

“…We generated a novel CRISPR library and performed the first genome-wide CRISPR screen with SARS-CoV-2. The identification of known pro-viral genes ACE2 and CTSL demonstrate the technical quality of the screen, providing confidence in the additional genes that regulate SARS-CoV-2 infection ( 8 , 14 , 15 , 44 ). We discovered genes involved in diverse biological processes including chromatin remodeling, histone modification, cellular signaling, and RNA regulation.…”

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

“…We performed the first genome-wide screens for host genes that affect infection with the pandemic CoVs SARS-CoV-2 and MERS-CoV as well as the recombinant bat CoV HKU5-SARS-CoV-1-S. Identification of the viral receptors ACE2 and DPP4 and the protease CTSL demonstrates the technical quality of the screens, providing confidence in the additional genes that regulate SARS-CoV-2 infection (Hoffmann et al, 2019(Hoffmann et al, , 2020bOu et al, 2020;Shang et al, 2020). We discovered genes involved in diverse biological processes, including chromatin remodeling, histone modification, cellular signaling, and RNA regulation.…”

“…In addition, we utilized a replication-competent infectious clone of SARS-CoV-2 expressing the fluorescent reporter mNeonGreen (icSARS-CoV-2-mNG) to quantify the influence of these molecules on viral replication (Xie et al, 2020). We observed dose-dependent inhibition of SARS-CoV-2-induced cell death and virus replication with calpain inhibitor III, whose targets include Cathepsin L (Figures 5A, 5D, and 5E; Simmons et al, 2005;Hoffmann et al, 2019). Given the pro-viral role of SMARCA4 and SMAD3, we tested whether existing small-molecule antagonists of these pathways have antiviral activity against SARS-CoV-2.…”

Faculty Opinions recommendation of Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

“…We performed the first genome-wide screens for host genes that affect infection with the pandemic CoVs SARS-CoV-2 and MERS-CoV as well as the recombinant bat CoV HKU5-SARS-CoV-1-S. Identification of the viral receptors ACE2 and DPP4 and the protease CTSL demonstrates the technical quality of the screens, providing confidence in the additional genes that regulate SARS-CoV-2 infection (Hoffmann et al, 2019(Hoffmann et al, , 2020bOu et al, 2020;Shang et al, 2020). We discovered genes involved in diverse biological processes, including chromatin remodeling, histone modification, cellular signaling, and RNA regulation.…”