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 Mutations in the Spike Protein of Middle East Respiratory Syndrome Coronavirus Transmitted in Korea Increase Resistance to Antibody-Mediated Neutralization

2018 ◽  
Vol 93 (2) ◽  
Author(s):  
Hannah Kleine-Weber ◽  
Mahmoud Tarek Elzayat ◽  
Lingshu Wang ◽  
Barney S. Graham ◽  
Marcel A. Müller ◽  
...  
Keyword(s):  
Middle East ◽  
Protein Binding ◽  
Receptor Binding ◽  
Viral Entry ◽  
Middle East Respiratory Syndrome ◽  
Spike Protein ◽  
Target Cells ◽  
S Protein ◽  
Hospital Outbreak ◽  
The Republic

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) poses a threat to public health. The virus is endemic in the Middle East but can be transmitted to other countries by travel activity. The introduction of MERS-CoV into the Republic of Korea by an infected traveler resulted in a hospital outbreak of MERS that entailed 186 cases and 38 deaths. The MERS-CoV spike (S) protein binds to the cellular protein DPP4 via its receptor binding domain (RBD) and mediates viral entry into target cells. During the MERS outbreak in Korea, emergence and spread of viral variants that harbored mutations in the RBD, D510G and I529T, was observed. Counterintuitively, these mutations were found to reduce DPP4 binding and viral entry into target cells. In this study, we investigated whether they also exerted proviral effects. We confirm that changes D510G and I529T reduce S protein binding to DPP4 but show that this reduction only translates into diminished viral entry when expression of DPP4 on target cells is low. Neither mutation modulated S protein binding to sialic acids, S protein activation by host cell proteases, or inhibition of S protein-driven entry by interferon-induced transmembrane proteins. In contrast, changes D510G and I529T increased resistance of S protein-driven entry to neutralization by monoclonal antibodies and sera from MERS patients. These findings indicate that MERS-CoV variants with reduced neutralization sensitivity were transmitted during the Korean outbreak and that the responsible mutations were compatible with robust infection of cells expressing high levels of DPP4. IMPORTANCE MERS-CoV has pandemic potential, and it is important to identify mutations in viral proteins that might augment viral spread. In the course of a large hospital outbreak of MERS in the Republic of Korea in 2015, the spread of a viral variant that contained mutations in the viral spike protein was observed. These mutations were found to reduce receptor binding and viral infectivity. However, it remained unclear whether they also exerted proviral effects. We demonstrate that these mutations reduce sensitivity to antibody-mediated neutralization and are compatible with robust infection of target cells expressing large amounts of the viral receptor DPP4.

scholarly journals Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2

2021 ◽  
Vol 17 (1) ◽  
pp. e1009212 ◽  
Author(s):  
Tianling Ou ◽  
Huihui Mou ◽  
Lizhou Zhang ◽  
Amrita Ojha ◽  
Hyeryun Choe ◽  
...  
Keyword(s):  
Viral Entry ◽  
Cathepsin L ◽  
Mechanistic Explanation ◽  
Furin Cleavage ◽  
Culture Studies ◽  
S Protein ◽  
Furin Cleavage Site ◽  
Endosomal Acidification ◽  
S Proteins

Hydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furin-cleavage site of the SARS-CoV-2 S protein is ablated or when it is introduced into the SARS-CoV-1 S protein. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19.

scholarly journals Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2

2020 ◽  
Author(s):  
Tianling Ou ◽  
Huihui Mou ◽  
Lizhou Zhang ◽  
Amrita Ojha ◽  
Hyeryun Choe ◽  
...  
Keyword(s):  
Cell Culture ◽  
Clinical Trials ◽  
Viral Infection ◽  
Viral Entry ◽  
Cleavage Site ◽  
Cathepsin L ◽  
Furin Cleavage ◽  
S Protein ◽  
Furin Cleavage Site

AbstractHydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furin-cleavage site of the SARS-CoV-2 S protein is ablated. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19.Author SummaryThe novel pathogenic coronavirus SARS-CoV-2 causes COVID-19 and remains a threat to global public health. Chloroquine and hydroxychloroquine have been shown to prevent viral infection in cell-culture systems, but human clinical trials did not observe a significant improvement in COVID-19 patients treated with these compounds. Here we show that hydroxychloroquine interferes with only one of two somewhat redundant pathways by which the SARS-CoV-2 spike (S) protein is activated to mediate infection. The first pathway is dependent on the endosomal protease cathepsin L and sensitive to hydroxychloroquine, whereas the second pathway is dependent on TMPRSS2, which is unaffected by this compound. We further show that SARS-CoV-2 is more reliant than SARS coronavirus (SARS-CoV-1) on the TMPRSS2 pathway, and that this difference is due to a furin cleavage site present in the SARS-CoV-2 S protein. Finally, we show that combinations of hydroxychloroquine and a clinically tested TMPRSS2 inhibitor work together to effectively inhibit SARS-CoV-2 entry. Thus TMPRSS2 expression on physiologically relevant SARS-CoV-2 target cells may bypass the antiviral activities of hydroxychloroquine, and explain its lack of in vivo efficacy.

scholarly journals Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay

2016 ◽  
Vol 60 (11) ◽  
pp. 6532-6539 ◽  
Author(s):  
Mizuki Yamamoto ◽  
Shutoku Matsuyama ◽  
Xiao Li ◽  
Makoto Takeda ◽  
Yasushi Kawaguchi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Middle East ◽  
Membrane Fusion ◽  
Serine Protease ◽  
Potent Inhibitor ◽  
Middle East Respiratory Syndrome ◽  
Target Cells ◽  
Dependent Manner ◽  
S Protein

ABSTRACTMiddle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS coronavirus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus, and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor-binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dependent manner using cell lines expressingRenillaluciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1,000 drugs approved for use by the U.S. Food and Drug Administration. Nafamostat also blocked MERS-CoV infectionin vitro. Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.

scholarly journals Structure, function and antigenicity of the SARS-CoV-2 spike glycoprotein

2020 ◽  
Author(s):  
Alexandra C. Walls ◽  
Young-Jun Park ◽  
M. Alexandra Tortorici ◽  
Abigail Wall ◽  
Andrew T. McGuire ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Target Cells ◽  
Furin Cleavage ◽  
Humoral Immune ◽  
Binding Domains ◽  
Furin Cleavage Site ◽  
Recent Emergence ◽  
Novel Coronavirus

SUMMARYThe recent emergence of a novel coronavirus associated with an ongoing outbreak of pneumonia (Covid-2019) resulted in infections of more than 72,000 people and claimed over 1,800 lives. Coronavirus spike (S) glycoprotein trimers promote entry into cells and are the main target of the humoral immune response. We show here that SARS-CoV-2 S mediates entry in VeroE6 cells and in BHK cells transiently transfected with human ACE2, establishing ACE2 as a functional receptor for this novel coronavirus. We further demonstrate that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, which correlates with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and other SARS-related CoVs. We determined a cryo-electron microscopy structure of the SARS-CoV-2 S ectodomain trimer, demonstrating spontaneous opening of the receptor-binding domain, and providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal sera potently inhibited SARS-CoV-2 S-mediated entry into target cells, thereby indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.

Interfering with Host Proteases in SARS-CoV-2 Entry as a Promising Therapeutic Strategy

2021 ◽  
Vol 28 ◽  
Author(s):  
Patrick Müller ◽  
Hannah Maus ◽  
Stefan Josef Hammerschmidt ◽  
Philip Knaff ◽  
Volker Mailänder ◽  
...  
Keyword(s):  
Cathepsin L ◽  
Cell Entry ◽  
New Drugs ◽  
Host Cells ◽  
S Protein ◽  
Cell Receptors ◽  
Current State ◽  
Causative Drug ◽  
Transmembrane Serine Protease ◽  
Global Threat

: Due to its fast international spread and substantial mortality, the coronavirus disease COVID-19 evolved to a global threat. Since currently, there is no causative drug against this viral infection available, science is striving for new drugs and approaches to treat the new disease. Studies have shown that the cell entry of coronaviruses into host cells takes place through the binding of the viral spike (S) protein to cell receptors. Priming of the S protein occurs via hydrolysis by different host proteases. The inhibition of these proteases could impair the processing of the S protein, thereby affecting the interaction with the host-cell receptors and preventing virus cell entry. Hence, inhibition of these proteases could be a promising strategy for treatment against SARS-CoV-2. In this review, we discuss the current state of the art of developing inhibitors against the entry proteases furin, the transmembrane serine protease type-II (TMPRSS2), trypsin, and cathepsin L.

scholarly journals CD81 Is Required for Hepatitis C Virus Glycoprotein-Mediated Viral Infection

2004 ◽  
Vol 78 (3) ◽  
pp. 1448-1455 ◽  
Author(s):  
Jie Zhang ◽  
Glenn Randall ◽  
Adrian Higginbottom ◽  
Peter Monk ◽  
Charles M. Rice ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Viral Entry ◽  
Human Liver ◽  
Cell Entry ◽  
Target Cells ◽  
Small Interfering Rnas ◽  
Efficient Cell Culture System ◽  
Human Liver Cell

ABSTRACT CD81 has been described as a putative receptor for hepatitis C virus (HCV); however, its role in HCV cell entry has not been characterized due to the lack of an efficient cell culture system. We have examined the role of CD81 in HCV glycoprotein-dependent entry by using a recently developed retroviral pseudotyping system. Human immunodeficiency virus (HIV) pseudotypes bearing HCV E1E2 glycoproteins show a restricted tropism for human liver cell lines. Although all of the permissive cell lines express CD81, CD81 expression alone is not sufficient to allow viral entry. CD81 is required for HIV-HCV pseudotype infection since (i) a monoclonal antibody specific for CD81 inhibited infection of susceptible target cells and (ii) silencing of CD81 expression in Huh-7.5 hepatoma cells by small interfering RNAs inhibited HIV-HCV pseudotype infection. Furthermore, expression of CD81 in human liver cells that were previously resistant to infection, HepG2 and HH29, conferred permissivity of HCV pseudotype infection. The characterization of chimeric CD9/CD81 molecules confirmed that the large extracellular loop of CD81 is a determinant for viral entry. These data suggest a functional role for CD81 as a coreceptor for HCV glycoprotein-dependent viral cell entry.

scholarly journals Epitope‐based peptide vaccine design and target site depiction against Middle East Respiratory Syndrome Coronavirus: an immune-informatics study

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Muhammad Tahir ul Qamar ◽  
Saman Saleem ◽  
Usman Ali Ashfaq ◽  
Amna Bari ◽  
Farooq Anwar ◽  
...  
Keyword(s):  
T Cell ◽  
Middle East ◽  
B Cell ◽  
Mhc Class Ii ◽  
Peptide Vaccine ◽  
Middle East Respiratory Syndrome ◽  
T Cell Epitopes ◽  
Resistance Response ◽  
B Cell Epitopes ◽  
S Protein

Abstract Background Middle East Respiratory Syndrome Coronavirus (MERS-COV) is the main cause of lung and kidney infections in developing countries such as Saudi Arabia and South Korea. This infectious single-stranded, positive (+) sense RNA virus enters the host by binding to dipeptidyl-peptide receptors. Since no vaccine is yet available for MERS-COV, rapid case identification, isolation, and infection prevention strategies must be used to combat the spreading of MERS-COV infection. Additionally, there is a desperate need for vaccines and antiviral strategies. Methods The present study used immuno-informatics and computational approaches to identify conserved B- and T cell epitopes for the MERS-COV spike (S) protein that may perform a significant role in eliciting the resistance response to MERS-COV infection. Results Many conserved cytotoxic T-lymphocyte epitopes and discontinuous and linear B-cell epitopes were predicted for the MERS-COV S protein, and their antigenicity and interactions with the human leukocyte antigen (HLA) B7 allele were estimated. Among B-cell epitopes, QLQMGFGITVQYGT displayed the highest antigenicity-score, and was immensely immunogenic. Among T-cell epitopes, MHC class-I peptide YKLQPLTFL and MHC class-II peptide YCILEPRSG were identified as highly antigenic. Furthermore, docking analyses revealed that the predicted peptides engaged in strong bonding with the HLA-B7 allele. Conclusion The present study identified several MERS-COV S protein epitopes that are conserved among various isolates from different countries. The putative antigenic epitopes may prove effective as novel vaccines for eradication and combating of MERS-COV infection.

scholarly journals Porcine deltacoronavirus enters cells via two pathways: A protease-mediated one at the cell surface and another facilitated by cathepsins in the endosome

2019 ◽  
Vol 294 (25) ◽  
pp. 9830-9843 ◽  
Author(s):  
Jialin Zhang ◽  
Jianfei Chen ◽  
Da Shi ◽  
Hongyan Shi ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Cultures ◽  
Viral Entry ◽  
Cathepsin B ◽  
Cathepsin L ◽  
The United States ◽  
Protein Cleavage ◽  
S Protein ◽  
Endosomal Pathway

Porcine deltacoronavirus (PDCoV) is a pathogen belonging to the genus Deltacoronavirus that in 2014 caused outbreaks of piglet diarrhea in the United States. To identify suitable therapeutic targets, a more comprehensive understanding of the viral entry pathway is required, particularly of the role of proteases. Here, we identified the proteases that activate the viral spike (S) glycoprotein to initiate cell entry and also pinpointed the host-cellular pathways that PDCoV uses for entry. Our results revealed that cathepsin L (CTSL) and cathepsin B (CTSB) in lysosomes and extracellular trypsin in cell cultures independently activate the S protein for membrane fusion. Pretreating the cells with the lysosomal acidification inhibitor bafilomycin-A1 (Baf-A1) completely inhibited PDCoV entry, and siRNA-mediated ablation of CTSL or CTSB expression significantly reduced viral infection, indicating that PDCoV uses an endosomal pathway for entry. Of note, trypsin treatment of cell cultures also activated PDCoV entry, even when the endosomal pathway was inhibited. This observation indicated that trypsin-induced S protein cleavage and activation in cell cultures enables viral entry directly from the cell surface. Our results provide critical insights into the PDCoV infection mechanism, uncovering two distinct viral entry pathways: one through cathepsin L and cathepsin B in the endosome and another via a protease at the cell surface. Because PDCoV infection sites represent a proteases-rich environment, these findings suggest that endosome inhibitor treatment alone is insufficient to block PDCoV entry into intestinal epithelial cells in vivo. Therefore, approaches that inhibit viral entry from the cell membrane should also be considered.

Abstract 12928: Vascular Complications in Covid-19 and Role of Zinc in Viral Inhibition

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Donghui Zhu ◽  
Zhen Zhao
Keyword(s):  
Viral Entry ◽  
Vascular Complications ◽  
Functional Change ◽  
Cell Entry ◽  
Host Cells ◽  
Binding Motif ◽  
Vascular Cells ◽  
Viral Inhibition ◽  
S Protein

Although COVID-19 is associated with severe respiratory dysfunctions, conspicuous vascular complications and neurological manifestations have been reported worldwide. Of note, two distinctive features have been noticed in severe patients, progressive increase of inflammation and an unusual trend of hypercoagulation. Interestingly, evidence is mounting that healthy blood vessels protect children from serious effects of COVID-19, such as stroke. These findings suggest vascular complications play a key role in the progress of COVID-19, warranting an investigation to its pathophysiology and treatment strategy related to vascular cells. Cell entry of this SARS-CoV-2 virus depends on binding of the viral spike (S) proteins to cellular receptor ACE2, which could be a key target for blocking the viral entry into host cells. ACE2 is a zinc (Zn) binding metallopeptidase while Zn possesses distinct antiviral properties against many human viruses including coronaviruses. Although the mechanistic studies are lacking, Zn appears to inhibit viral protease and polymerase enzymatic processes, and physical processes such as virus attachment, cell entry, and uncoating. In fact, our data showed that ACE2 has multiple affinity binding sites for Zn. Excess bindings of ionic Zn to ACE2 led to its conformational or functional change, therefore, interfering with its ability to metabolize its substrate as well as inhibiting its binding to S protein. Computational modeling also revealed that one critical Zn binding motif is located in ACE2’s binding domain to S protein, and docking affinity of S protein to ACE2 was significantly reduced after Zn binding to this specific site. Moreover, cell and animal studies using pseudo-virus bearing CoV-2-S protein validated that significantly lower infection of vascular cells in the presence of Zn was observed. Thus, targeting vascular complications in COVID-19 may offer strong benefits including the potential therapeutic role of Zn.

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