scholarly journals Middle East Respiratory Syndrome Coronavirus Infection Mediated by the Transmembrane Serine Protease TMPRSS2

2013 ◽  
Vol 87 (23) ◽  
pp. 12552-12561 ◽  
Author(s):  
Kazuya Shirato ◽  
Miyuki Kawase ◽  
Shutoku Matsuyama
Keyword(s):  
Middle East ◽  
Cell Surface ◽  
Serine Protease ◽  
Virus Entry ◽  
Cathepsin L ◽  
Vero Cells ◽  
Middle East Respiratory Syndrome ◽  
Simultaneous Treatment ◽  
Virus Growth ◽  
Transmembrane Serine Protease

The Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host proteases for virus entry into lung cells. In the current study, Vero cells constitutively expressing type II transmembrane serine protease (Vero-TMPRSS2 cells) showed larger syncytia at 18 h after infection with MERS-CoV than after infection with other coronaviruses. Furthermore, the susceptibility of Vero-TMPRSS2 cells to MERS-CoV was 100-fold higher than that of non-TMPRSS2-expressing parental Vero cells. The serine protease inhibitor camostat, which inhibits TMPRSS2 activity, completely blocked syncytium formation but only partially blocked virus entry into Vero-TMPRSS2 cells. Importantly, the coronavirus is thought to enter cells via two distinct pathways, one mediated by TMPRSS2 at the cell surface and the other mediated by cathepsin L in the endosome. Simultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry into Vero-TMPRSS2 cells, indicating that MERS-CoV employs both the cell surface and the endosomal pathway to infect Vero-TMPRSS2 cells. In contrast, a single camostat treatment suppressed MERS-CoV entry into human bronchial submucosal gland-derived Calu-3 cells by 10-fold and virus growth by 270-fold, although treatment with both camostat and (23,25)-trans-epoxysuccinyl-l-leucylamindo-3-methylbutane ethyl ester, a cathepsin inhibitor, or treatment with leupeptin, an inhibitor of cysteine, serine, and threonine peptidases, was no more efficacious than treatment with camostat alone. Further, these inhibitors were not efficacious against MERS-CoV infection of MRC-5 and WI-38 cells, which were derived from lung, but these characters differed from those of mature pneumocytes. These results suggest that a single treatment with camostat is sufficient to block MERS-CoV entry into a well-differentiated lung-derived cell line.

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

2018 ◽  
Vol 92 (19) ◽  
Author(s):  
Shutoku Matsuyama ◽  
Kazuya Shirato ◽  
Miyuki Kawase ◽  
Yutaka Terada ◽  
Kengo Kawachi ◽  
...  
Keyword(s):  
Middle East ◽  
Viral Entry ◽  
Cathepsin L ◽  
Inhibitory Effect ◽  
Middle East Respiratory Syndrome ◽  
Cell Entry ◽  
Target Cells ◽  
Furin Cleavage ◽  
S Protein ◽  
Entry Assay

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.

scholarly journals Infectious Middle East Respiratory Syndrome Coronavirus Excretion and Serotype Variability Based on Live Virus Isolates from Patients in Saudi Arabia

2015 ◽  
Vol 53 (9) ◽  
pp. 2951-2955 ◽  
Author(s):  
Doreen Muth ◽  
Victor M. Corman ◽  
Benjamin Meyer ◽  
Abdullah Assiri ◽  
Malak Al-Masri ◽  
...  
Keyword(s):  
Middle East ◽  
Infectious Virus ◽  
Virus Isolation ◽  
Vero Cells ◽  
Prototype Strain ◽  
Middle East Respiratory Syndrome ◽  
Serum Samples ◽  
Live Virus ◽  
Virus Isolates ◽  
Rna Concentration

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 1,082 people, including 439 fatalities. So far, no empirical virus isolation study has been done to elucidate infectious virus secretion or serotype variability. Here, we used 51 respiratory samples from 32 patients with confirmed MERS-CoV infection for virus isolation in Vero B4 and Caco-2 cells. We found Caco-2 cells to significantly enhance isolation success over routinely used Vero cells. Isolation success correlated with viral RNA concentration and time after diagnosis as well as with the amount of IgA antibodies secreted in respiratory samples used for isolation. Results from plaque reduction neutralization assays using a representative range of serum samples and virus isolates suggested that all circulating human MERS-CoV strains represent one single serotype. The choice of prototype strain is not likely to influence the success of candidate MERS-CoV vaccines. However, vaccine formulations should be evaluated for their potential to induce IgA.

scholarly journals Vaccinia Virus Envelope D8L Protein Binds to Cell Surface Chondroitin Sulfate and Mediates the Adsorption of Intracellular Mature Virions to Cells

1999 ◽  
Vol 73 (10) ◽  
pp. 8750-8761 ◽  
Author(s):  
Jye-Chian Hsiao ◽  
Che-Sheng Chung ◽  
Wen Chang
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Chondroitin Sulfate ◽  
Virus Entry ◽  
Viral Envelope ◽  
Binding Assays ◽  
Viral Envelope Protein ◽  
Virus Envelope ◽  
Virus Growth ◽  
Control Virus

ABSTRACT We previously showed that an envelope A27L protein of intracellular mature virions (IMV) of vaccinia virus binds to cell surface heparan sulfate during virus infection. In the present study we identified another viral envelope protein, D8L, that binds to chondroitin sulfate on cells. Soluble D8L protein interferes with the adsorption of wild-type vaccinia virions to cells, indicating a role in virus entry. To explore the interaction of cell surface glycosaminoglycans and vaccinia virus, we generated mutant viruses from a control virus, WR32-7/Ind14K (A27L+ D8L+) to be defective in expression of either the A27L or the D8L gene (A27L+D8L− or A27L− D8L+) or both (A27L− D8L−). The A27L+D8L+ and A27L− D8L+ mutants grew well in BSC40 cells, consistent with previous observations. However, the IMV titers of A27L+ D8L− and A27L− D8L− viruses in BSC40 cells were reduced, reaching only 10% of the level for the control virus. The data suggested an important role for D8L protein in WR32-7/Ind14K virus growth in cell cultures. A27L protein, on the other hand, could not complement the functions of D8L protein. The low titers of the A27L+ D8L− and A27L−D8L− mutant viruses were not due to defects in the morphogenesis of IMV, and the mutant virions demonstrated a brick shape similar to that of the control virions. Furthermore, the infectivities of the A27L+ D8L− and A27L−D8L− mutant virions were 6 to 10% of that of the A27L+ D8L+ control virus. Virion binding assays revealed that A27L+ D8L− and A27L− D8L− mutant virions bound less well to BSC40 cells, indicating that binding of viral D8L protein to cell surface chondroitin sulfate could be important for vaccinia virus entry.

scholarly journals Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein

2017 ◽  
Vol 114 (40) ◽  
pp. E8508-E8517 ◽  
Author(s):  
Wentao Li ◽  
Ruben J. G. Hulswit ◽  
Ivy Widjaja ◽  
V. Stalin Raj ◽  
Ryan McBride ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Middle East ◽  
Cell Surface ◽  
Human Erythrocytes ◽  
Middle East Respiratory Syndrome ◽  
Host Cells ◽  
Dromedary Camel ◽  
Differential Distribution ◽  
Neuraminidase Treatment ◽  
Intact Virus

Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract both in humans and in its natural host, the dromedary camel. Virion attachment to host cells is mediated by 20-nm-long homotrimers of spike envelope protein S. The N-terminal subunit of each S protomer, called S1, folds into four distinct domains designated S1A through S1D. Binding of MERS-CoV to the cell surface entry receptor dipeptidyl peptidase 4 (DPP4) occurs via S1B. We now demonstrate that in addition to DPP4, MERS-CoV binds to sialic acid (Sia). Initially demonstrated by hemagglutination assay with human erythrocytes and intact virus, MERS-CoV Sia-binding activity was assigned to S subdomain S1A. When multivalently displayed on nanoparticles, S1 or S1A bound to human erythrocytes and to human mucin in a strictly Sia-dependent fashion. Glycan array analysis revealed a preference for α2,3-linked Sias over α2,6-linked Sias, which correlates with the differential distribution of α2,3-linked Sias and the predominant sites of MERS-CoV replication in the upper and lower respiratory tracts of camels and humans, respectively. Binding is hampered by Sia modifications such as 5-N-glycolylation and (7,)9-O-acetylation. Depletion of cell surface Sia by neuraminidase treatment inhibited MERS-CoV entry of Calu-3 human airway cells, thus providing direct evidence that virus–Sia interactions may aid in virion attachment. The combined observations lead us to propose that high-specificity, low-affinity attachment of MERS-CoV to sialoglycans during the preattachment or early attachment phase may form another determinant governing the host range and tissue tropism of this zoonotic pathogen.

Metalloprotease meprin β is activated by transmembrane serine protease matriptase-2 at the cell surface thereby enhancing APP shedding

2015 ◽  
Vol 470 (1) ◽  
pp. 91-103 ◽  
Author(s):  
Felix Jäckle ◽  
Frederike Schmidt ◽  
Rielana Wichert ◽  
Philipp Arnold ◽  
Johannes Prox ◽  
...  
Keyword(s):  
Cell Surface ◽  
Serine Protease ◽  
Transmembrane Proteins ◽  
Mechanistic Insight ◽  
Transmembrane Serine Protease ◽  
Proenzyme Activation

Metalloprotease meprin β is a sheddase of transmembrane proteins. We identified serine protease matriptase-2 (MT2) as a specific activator of meprin β at the cell surface. This provides mechanistic insight for the regulation of meprin β activity and demonstrates clear differences in proenzyme activation.

scholarly journals A Transmembrane Serine Protease Is Linked to the Severe Acute Respiratory Syndrome Coronavirus Receptor and Activates Virus Entry

2010 ◽  
Vol 85 (2) ◽  
pp. 873-882 ◽  
Author(s):  
A. Shulla ◽  
T. Heald-Sargent ◽  
G. Subramanya ◽  
J. Zhao ◽  
S. Perlman ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Serine Protease ◽  
Virus Entry ◽  
Transmembrane Serine Protease

scholarly journals Aerosolized type II transmembrane serine protease 2 inhibitor to combat COVID-19: a proposal

2021 ◽  
Vol 8 (12) ◽  
pp. 1996
Author(s):  
Chandan Raybarman ◽  
Surajit Bhattacharjee
Keyword(s):  
Cell Surface ◽  
Serine Protease ◽  
Viral Entry ◽  
Type Ii ◽  
Cellular Entry ◽  
Transmembrane Serine Protease

Type II transmembrane serine protease (TMPRSS2) is expressed at the cell surface with COVID-19 infection. And COVID-19 infection misuse TMPRSS2 to advance their spread, making this protease potential focuses for intervention in COVID-19 infection. TMPRSS2 blocker may be the appropriate option to arrest cellular entry of COVID-19 by deregulating spike priming. Therefore a trial may be intended to watch the adequacy of aerosolized spraying of TMPRSS2 inhibitors to break the viral entry to the objective cells that empower to break the COVID-19 transmission. Targeting TMPRSS2 through aerosolized TMPRSS2 inhibitor is important to examine a possibly viable remedial technique in the treatment of COVID-19.

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