Novel proteolytic activation of Ebolavirus glycoprotein GP by TMPRSS2 and cathepsin L at an uncharted position can compensate for furin cleavage

• Published in: Virus research Vol. 347; p. 199430
• Main Authors: Bestle, Dorothea, Bittel, Linda, Werner, Anke-Dorothee, Kämper, Lennart, Dolnik, Olga, Krähling, Verena, Steinmetzer, Torsten, Böttcher-Friebertshäuser, Eva
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-09-2024; Elsevier
• Subjects: Animals; Cathepsin L - genetics; Cathepsin L - metabolism; Cell Line; Chlorocebus aethiops; Ebola virus; Ebolavirus - genetics; Ebolavirus - metabolism; Ebolavirus - physiology; Endosomal cathepsins; Endosomes - metabolism; Endosomes - virology; Furin; Furin - genetics; Furin - metabolism; GP cleavage; Humans; Proteolysis; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; TMPRSS2; Vero Cells; Viral Envelope Proteins - genetics; Viral Envelope Proteins - metabolism; Virus Internalization; Ebola virus; Furin; TMPRSS2; Endosomal cathepsins; GP cleavage
• ISSN: 0168-1702; 1872-7492; 1872-7492
• DOI: 10.1016/j.virusres.2024.199430

•The necessity of EBOV GP cleavage at the furin cleavage site was and is a subject of debate.•The furin cleavage site mutant EBOV GP_AGTAA, which was described as non-cleavable, is shown to be cleaved by TMPRSS2 and cathepsin L.•IF suggests that TMPRSS2 may cleave EBOV GP upon entry in the late endosome or at later stages in the TGN.•Proteolytic activation of EBOV GP offers even greater flexibility than previously assumed.
A multistep priming process involving furin and endosomal cathepsin B and L (CatB/L) has been described for the Orthoebolavirus zairense (EBOV) glycoprotein GP. Inhibition or knockdown of either furin or endosomal cathepsins, however, did not prevent virus multiplication in cell cultures. Moreover, an EBOV mutant lacking the furin cleavage motif (RRTRR→AGTAA) was able to replicate and cause fatal disease in nonhuman primates, indicating that furin cleavage may be dispensable for virus infectivity. Here, by using protease inhibitors and EBOV GP-carrying recombinant vesicular stomatitis virus (VSV) and transcription and replication-competent virus-like particles (trVLPs) we found that processing of EBOV GP is mediated by different proteases in different cell lines depending on the protease repertoire available. Endosomal cathepsins were essential for EBOV GP entry in Huh-7 but not in Vero cells, in which trypsin-like proteases and stably expressed trypsin-like transmembrane serine protease 2 (TMPRSS2) supported wild-type EBOV GP and EBOV GP_AGTAA mutant entry. Furthermore, we show that the EBOV GP_AGTAA mutant is cleaved into fusion-competent GP2 by TMPRSS2 and by CatL at a so far unknown site. Fluorescence microscopy co-localization studies indicate that EBOV GP cleavage by TMPRSS2 may occur in the TGN prior to virus release or in the late endosome at the stage of virus entry into a new cell.
Our data show that EBOV GP must be proteolytically activated to support virus entry but has even greater flexibility in terms of proteases and the precise cleavage site than previously assumed.