TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells.

Dorothea Bestle,Hong Moulton,Olga Dolnik,Wolfgang Garten,Hans-Dieter Klenk,Markus Eickmann,Miriam Ruth Heindl,Oliver Pilgram,Torsten Steinmetzer,Eva Böttcher-Friebertshäuser,Cornelius Rohde,Hannah Limburg,Thuy Van Lam Van,Kornelia Hardes,David A Stein

doi:10.26508/lsa.202000786

Abstract

The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2' site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.

Highlights

In December 2019, a new coronavirus (CoV) emerged and has rapidly spread around the world causing a pandemic never before observed with these viruses
The analogous sequences of the S proteins from Middle East respiratory syndrome (MERS)-CoV, severe acute respiratory syndrome (SARS)-CoV, and avian infectious bronchitis virus (IBV) strain Beaudette were prepared as reference substrates
Sequence analyses of the S protein of the emerged SARS-CoV-2 suggested that the R-R-A-R motif at the S1/S2 site may be sensitive to cleavage by furin, whereas the S29 site contains a single R residue that can be cleaved by trypsin-like serine proteases such as TMPRSS2 [20, 22, 23]

Summary

Introduction

In December 2019, a new coronavirus (CoV) emerged and has rapidly spread around the world causing a pandemic never before observed with these viruses. More recent studies by us and others demonstrated that TMPRSS2-deficient mice do not suffer from pathology when infected with certain influenza A virus strains, SARS-CoV and MERSCoV due to inhibition of proteolytic activation of progeny virus and inhibition of virus spread along the respiratory tract [15, 16, 17, 18]. Pretreatment of human Caco-2 colon and human airway cells with the broad range inhibitor camostat mesylate, which can inhibit TMPRSS2 activity, markedly reduced the entry of SARS-CoV-2 as well as vesicular stomatitis virus pseudotypes containing the SARS-CoV-2 S protein This suggests that a trypsin-like serine protease is crucial for SARS-CoV-2 entry into these cells. Our results show that this approach has considerable therapeutic potential for treatment of COVID-19

Results

Discussion

Materials and Methods