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

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.

Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways.

Cleavage of influenza virus hemagglutinin (HA) by host proteases is essential for virus infectivity. HA of most influenza A and B (IAV/IBV) viruses is cleaved at a monobasic motif by trypsin-like proteases. Previous studies have reported that transmembrane serine protease 2 (TMPRSS2) is essential for activation of H7N9 and H1N1pdm IAV in mice but that H3N2 IAV and IBV activation is independent of TMPRSS2 and carried out by as-yet-undetermined protease(s). Here, to identify additional H3 IAV- and IBV-activating proteases, we used RNA-Seq to investigate the protease repertoire of murine lower airway tissues, primary type II alveolar epithelial cells (AECIIs), and the mouse lung cell line MLE-15. Among 13 candidates identified, TMPRSS4, TMPRSS13, hepsin, and prostasin activated H3 and IBV HA in vitro IBV activation and replication was reduced in AECIIs from Tmprss2/Tmprss4-deficient mice compared with WT or Tmprss2-deficient mice, indicating that murine TMPRSS4 is involved in IBV activation. Multicycle replication of H3N2 IAV and IBV in AECIIs of Tmprss2/Tmprss4-deficient mice varied in sensitivity to protease inhibitors, indicating that different, but overlapping, sets of murine proteases facilitate H3 and IBV HA cleavages. Interestingly, human hepsin and prostasin orthologs did not activate H3, but they did activate IBV HA in vitro Our results indicate that TMPRSS4 is an IBV-activating protease in murine AECIIs and suggest that TMPRSS13, hepsin, and prostasin cleave H3 and IBV HA in mice. They further show that hepsin and prostasin orthologs might contribute to the differences observed in TMPRSS2-independent activation of H3 in murine and human airways.