Proteolytic activation of Zaire ebolavirus and SARS-CoV-2 class I membrane fusion proteins by different host cell proteases

The proteolytic processing of viral fusion proteins by host cell proteases is a prerequisite for the replication and infectivity of enveloped viruses. The identification of relevant proteases involved in virus activation is therefore an important step towards the development of host protease inhibit...

Full description

Saved in:
Bibliographic Details
Main Author: Dorothea, Bestle
Contributors: Friebertshäuser, Eva (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Language:English
Published: Philipps-Universität Marburg 2022
Subjects:
Online Access:PDF Full Text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The proteolytic processing of viral fusion proteins by host cell proteases is a prerequisite for the replication and infectivity of enveloped viruses. The identification of relevant proteases involved in virus activation is therefore an important step towards the development of host protease inhibitors as antiviral strategies. In this study the proteolytic activation mechanisms of the class I fusion proteins of Zaire ebolavirus (ZEBOV) and the novel severe acute respiratory syndrome (SARS) coronavirus (CoV) 2, which both possess two cleavage sites, were investigated. The fusion protein S of SARS-CoV-2 contains two distinct cleavage sites, with a multibasic motif at the S1/S2 site and a monobasic motif at the S2' site. Co-expression in 293F cells and infection studies in Calu-3 cells in the presence and absence of protease inhibitors revealed proteolytic activation by furin and transmembrane serine protease 2 (TMPRSS2). Both proteases have been shown to be essential for efficient replication of SARS-CoV-2, as they were not able to compensate for each other. In contrast, in vitro experiments revealed a highly complex processing mechanism for the ZEBOV GP beyond the described classical proteolytic activation by furin and endosomal cathepsin B and L. The "non-cleavable" ZEBOV GP_AGTAA furin cleavage mutant was shown to be processed into the 20 kDa fusion competent GP2 by TMPRSS2 and cathepsin L at so far unknown positions. Moreover, pseudovirus rVSV∆G ZEBOV GP growth kinetics and transcription and replication-competent virus-like particle (trVLP) entry studies in Huh-7, VeroE6 and Vero-TMPRSS2 cells in the presence and absence of protease inhibitors demonstrated, that protease-dependency of ZEBOV GP is determined by the protease repertoire of the respective cell line. Endosomal cathepsins were essential for the entry of ZEBOV GP into Huh-7 cells and combined treatment with inhibitors of furin (MI-1148), cathepsins (E64d) or TMPRSS2 (BAPA or MI-432) was able to completely abolish rVSV∆G ZEBOV GP replication. In contrast, this was not the case for VeroE6 and Vero-TMPRSS2 cells, indicating that ZEBOV GP is able to use other so far unknown protease(s) for the proteolytic processing in Vero cells. Host cell protease cleavage seems to be an essential mechanism for ZEBOV GP replication, as the generation of highly truncated GP variants resulted in either still cleavable or functionally inactive GP. Additionally the subcellular localization of the host cell protease TMPRSS2, involved in the processing of fusion proteins of many respiratory viruses, including influenza virus HA and SARS-CoV-2 S, was analyzed in Huh-7 cells. TMPRSS2 has been found to accumulate as previously described in the TGN but was furthermore found in the ERGIC and to a certain extent in the recycling and late endosome of TMPRSS2-overexpressing Huh-7 cells.
DOI:10.17192/z2023.0022