Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin. Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.

Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Spike Protein Proteolytic Processing

“…Interestingly, coronavirus spike proteins and in particular the envelope glycoprotein of Middle East respiratory syndrome coronavirus (MERS-CoV) have been shown in cell culture to be cleaved by a number of host cell proteases such as cathepsins [12][13][14], trypsin-like proteases, notably membrane-bound transmembrane protease, serine 2 (TMPRSS2) [12][13][14][15], and members of the pro-protein convertase family of enzymes such as furin [16,17]. The latter group of proteases may not be major players for typical routes of virus entry in respiratory epithelial cells [18], but could nonetheless be the S1 receptor binding domain and the S2 fusion machinery domain. S1/S2 denotes the proteolytic cleavage site between S1 and S2 domains.…”

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells

“…SARS-CoV and Middle East respiratory syndrome (MERS)-CoV can enter cells via endocytosis and use cathepsin in endosomes for activation (12)(13)(14). However, TMPRSS2 expression Data deposition: Data have been deposited in the Global Initiative on Sharing All Influenza Data (GISAID) database, https://www.gisaid.org/ (accession ID EPI_ISL_408667).…”

Zika virus: mapping and reprogramming the entry

“…One may conclude that furin or a furin-like enzyme activates progeny viruses, while no second protease is required during entry to susceptible cell (as reported for other members of Flaviviridae family) [60,61]. By contrast, some results advocating a role for furin during virus maturation may be due to an artifact, linked to the low specificity of the protease inhibitors used [73].…”