J Virol. 2018 Sep 12;92(19). pii: e00683-18. doi: 10.1128/JVI.00683-18. Print 2018 Oct 1.
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.
IMPORTANCE 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.
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.
IMPORTANCE 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.
Copyright © 2018 American Society for Microbiology.
KEYWORDS:
TMPRSS2; cathepsin L; coronavirus; dec-RVKR-CMK; furin- PMID:
- 30021905
- PMCID:
- PMC6146822
- DOI:
- 10.1128/JVI.00683-18
- [Indexed for MEDLINE]
Here we discuss the use of host proteases by MERS-CoV for cell entry. Studies show clearly that TMPRSS2 activates the MERS-CoV S protein (4, 10, 27). Expression of TMPRSS2 at the cell surface induces both virus entry into cells and cell-cell fusion of S protein-expressing cells (4, 10, 27). Furthermore, these phenomena are suppressed by the serine protease inhibitors camostat mesylate and nafamostat mesylate (4, 10, 28). In addition, other extracellular proteases, such as trypsin and elastase, activate the MERS-CoV S protein in a manner similar to that of TMPRSS2 (10).
. Also, our previous study shows that cathepsin L requires chlorpromazine, a membrane-permeable cationic drug that lowers the energy requirement for membrane fusion, to induce a small increase in cell entry by murine coronavirus (30).Therefore, it is unclear whether cathepsin L plays a role in activating the coronavirus S protein. However, it is at least certain that the enzymatic activity of cathepsin is necessary for proteolytic activation of the MERS-CoV cell entry because expression of TMPRSS2 overcomes blockade of viral cell entry by cathepsin inhibitors (10).
Taken together, the results presented herein do not support a role for cellular furin during direct activation of MERS-CoV S protein for viral cell entry. These findings are compatible with those of a previous study by Gierer et al. showing that MERS-CoV does not require furin for infectivity (14). Park et al. report that the only role played by furin during MERS-CoV infection is to determine the cell tropism of the virus (8). What is certain is that the MERS-CoV S protein precleaved by furin at the S1/S2 site still requires cell surface/extracellular and endosomal proteases, such as TMPRSS2, elastase, and cathepsin L, for cleavage at the S2′ site.
Schematic
illustration of the MERS-CoV spike glycoprotein and location of the
receptor binding domain (RBD) polymorphisms. The MERS-CoV spike
glycoprotein (MERS-S) consists of two subunits (S1 and S2). The S1
subunit contains an N-terminal signal peptide (SP) and an RBD, which
binds to the receptor DPP4. The S2 subunit harbors the functional
elements required for membrane fusion, a fusion peptide (FP), and two
heptad repeats (HR1 and HR2), as well as the transmembrane domain (TD)
and a cytoplasmic tail (CT). Below the scheme, the locations of the four
amino acid polymorphisms investigated in this study (L411F, F473S,
D510G, and I529T) are highlighted (bold letters).
https://www.ncbi.nlm.nih.gov/protein/YP_009047204.1
... , we conclude that the S protein is cleaved by furin during biogenesis, not on virions after they exit the cells.
... , we conclude that the S protein is cleaved by furin during biogenesis, not on virions after they exit the cells.
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