Roles of host proteases in the entry of SARS-CoV-2

Abstract

The spike protein (S) of SARS-CoV-2 is responsible for viral attachment and entry, thus a major factor for host susceptibility, tissue tropism, virulence and pathogenicity. The S is divided with S1 and S2 region, and the S1 contains the receptor-binding domain (RBD), while the S2 contains the hydrophobic fusion domain for the entry into the host cell. Numerous host proteases have been implicated in the activation of SARS-CoV-2 S through various cleavage sites. In this article, we review host proteases including furin, trypsin, transmembrane protease serine 2 (TMPRSS2) and cathepsins in the activation of SARS-CoV-2 S. Many betacoronaviruses including SARS-CoV-2 have polybasic residues at the S1/S2 site which is subjected to the cleavage by furin. The S1/S2 cleavage facilitates more assessable RBD to the receptor ACE2, and the binding triggers further conformational changes and exposure of the S2' site to proteases such as type II transmembrane serine proteases (TTPRs) including TMPRSS2. In the presence of TMPRSS2 on the target cells, SARS-CoV-2 can utilize a direct entry route by fusion of the viral envelope to the cellular membrane. In the absence of TMPRSS2, SARS-CoV-2 enter target cells via endosomes where multiple cathepsins cleave the S for the successful entry. Additional host proteases involved in the cleavage of the S were discussed. This article also includes roles of 3C-like protease inhibitors which have inhibitory activity against cathepsin L in the entry of SARS-CoV-2, and discussed the dual roles of such inhibitors in virus replication.

Fig. 1

Experimentally observed host protease cleavage sites in S of SARS-CoV-2, SARS-CoV and MERS-CoV. A. The S is divided into two portions: S1 and S2. S1 contains the receptor-binding domain (RBD) where the receptor-binding motif (RBM) interacts with the functional receptor (ACE2 or DPP4). The S2 domain contains the fusion domain, which is responsible for viral entry into the cell. In betacoronavirus entry studies, two cleavage sites have been observed as S1/S2 which is cleaved to cause a conformational change to facilitate binding to the functional receptor and S2’ which is cleaved to expose the fusion domain to allow for viral entry. The S2’ site is highly conserved among all coronaviruses and is cleaved by TMPRSS2. (a) In SARS-CoV-2, the S1/S2 cleavage site can be cleaved by host furin and trypsin, and two cathepsin L cleavage sites have been observed in S1 region as pictured. (b) SARS-CoV is unable to be cleaved by host furin but has been observed to be cleaved at the S1/S2 site experimentally by trypsin (Kim et al. 2022b). In addition to TMPRSS2 cleavage at S2’ site, human airway trypsin-like protease (HAT) has also been experimentally observed to have cleavage activity at the site. (c) MERS-CoV S is able to be cleaved by the same proteases as SARS-CoV-2 in the S1/S2 and S2’ sites. B. The multibasic arginine (R) resides observed in S of SARS-CoV-2 and MERS-CoV in the S1/S2 region allow for the cleavage of host furin, which shows a specificity for the motif RXXR. S2’ is highly conserved among coronaviruses, as it is responsible for exposing the fusion domain to allow for cell entry

Fig. 2

PACMAN prediction of SARS-CoV-2 cathepsin cleavage sites. A PACMAN predicted cleavage site of SARS-CoV-2 near S1/S2 and S2’ by cathepsins K, L, S and V aligned with other coronaviruses of importance. *Cathepsin L has also been predicted to cleave at this site in studies of MERS-CoV S. B Predicted cleavage site of the cathepsins near the S2’ cleavage site

Fig. 3

Attachment and entry model of SARS-CoV-2. When SARS-CoV-2 is released by the parental cell, some S is cleaved by host furin. Cleavage by furin facilitates faster binding to the functional receptor ACE2. The binding of ACE2 to S induces a conformational change which exposes the S2’ cleavage site. The presence or absence of TMPRSS2 dictates whether the virus enters through a fast membrane fusion or a slow endosomal route. In the absence of TMPRSS2, the virus is taken into an endosome where the pH will drop, activating cathepsin L. Cathepsin L cleaves S to initiate fusion to the endosomal membrane before release of viral RNA into the cytosol. In the presence of TMPRSS2, the S2’ site is cleaved and the virus can fuse directly to the cell membrane, allowing for a more rapid entry of the viral RNA into the cell