scholarly journals Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Kazuya Shirato ◽  
Kazuhiko Kanou ◽  
Miyuki Kawase ◽  
Shutoku Matsuyama
Keyword(s):  
Cell Surface ◽  
Cathepsin L ◽  
Laboratory Strain ◽  
Airway Epithelial Cells ◽  
Cell Entry ◽  
Clinical Isolates ◽  
Airway Epithelial ◽  
Human Coronavirus ◽  
S Protein ◽  
Human Coronavirus 229E

ABSTRACT Human coronavirus 229E (HCoV-229E), a causative agent of the common cold, enters host cells via two distinct pathways: one is mediated by cell surface proteases, particularly transmembrane protease serine 2 (TMPRSS2), and the other by endosomal cathepsin L. Thus, specific inhibitors of these proteases block virus infection. However, it is unclear which of these pathways is actually utilized by HCoV-229E in the human respiratory tract. Here, we examined the mechanism of cell entry used by a pseudotyped virus bearing the HCoV-229E spike (S) protein in the presence or absence of protease inhibitors. We found that, compared with a laboratory strain isolated in 1966 and passaged for a half century, clinical isolates of HCoV-229E were less likely to utilize cathepsin L; rather, they showed a preference for TMPRSS2. Two amino acid substitutions (R642M and N714K) in the S protein of HCoV-229E clinical isolates altered their sensitivity to a cathepsin L inhibitor, suggesting that these amino acids were responsible for cathepsin L use. After 20 passages in HeLa cells, the ability of the isolate to use cathepsin increased so that it was equal to that of the laboratory strain; this increase was caused by an amino acid substitution (I577S) in the S protein. The passaged virus showed a reduced ability to replicate in differentiated airway epithelial cells cultured at an air-liquid interface. These results suggest that the endosomal pathway is disadvantageous for HCoV-229E infection of human airway epithelial cells; therefore, clinical isolates are less able to use cathepsin. IMPORTANCE Many enveloped viruses enter cells through endocytosis. Viral spike proteins drive the fusion of viral and endosomal membranes to facilitate insertion of the viral genome into the cytoplasm. Human coronavirus 229E (HCoV-229E) utilizes endosomal cathepsin L to activate the spike protein after receptor binding. Here, we found that clinical isolates of HCoV-229E preferentially utilize the cell surface protease TMPRSS2 rather than endosomal cathepsin L. The endosome is a main site of Toll-like receptor recognition, which then triggers an innate immune response; therefore, HCoV-229E presumably evolved to bypass the endosome by entering the cell via TMPRSS2. Thus, the virus uses a simple mechanism to evade the host innate immune system. Therefore, therapeutic agents for coronavirus-mediated diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), should target cell surface TMPRSS2 rather than endosomal cathepsin.

scholarly journals Protease-Mediated Entry via the Endosome of Human Coronavirus 229E

2008 ◽  
Vol 83 (2) ◽  
pp. 712-721 ◽  
Author(s):  
Miyuki Kawase ◽  
Kazuya Shirato ◽  
Shutoku Matsuyama ◽  
Fumihiro Taguchi
Keyword(s):  
Cell Surface ◽  
Protease Inhibitors ◽  
Hepatitis Virus ◽  
Cathepsin L ◽  
Syncytium Formation ◽  
Human Coronavirus ◽  
S Protein ◽  
Mode Of Entry ◽  
Group I ◽  
Human Coronavirus 229E

ABSTRACT Human coronavirus 229E, classified as a group I coronavirus, utilizes human aminopeptidase N (APN) as a receptor; however, its entry mechanism has not yet been fully elucidated. We found that HeLa cells infected with 229E via APN formed syncytia when treated with trypsin or other proteases but not in a low-pH environment, a finding consistent with syncytium formation by severe acute respiratory syndrome coronavirus (SARS-CoV). In addition, trypsin induced cleavage of the 229E S protein. By using infectious viruses and pseudotyped viruses bearing the 229E S protein, we found that its infection was profoundly blocked by lysosomotropic agents as well as by protease inhibitors that also prevented infection with SARS-CoV but not that caused by murine coronavirus mouse hepatitis virus strain JHMV, which enters cells directly from the cell surface. We found that cathepsin L (CPL) inhibitors blocked 229E infection the most remarkably among a variety of protease inhibitors tested. Furthermore, 229E infection was inhibited in CPL knockdown cells by small interfering RNA, compared with what was seen for a normal counterpart producing CPL. However, its inhibition was not so remarkable as that found with SARS-CoV infection, which seems to indicate that while CPL is involved in the fusogenic activation of 229E S protein in endosomal infection, not-yet-identified proteases could also play a part in that activity. We also found 229E virion S protein to be cleaved by CPL. Furthermore, as with SARS-CoV, 229E entered cells directly from the cell surface when cell-attached viruses were treated with trypsin. These findings suggest that 229E takes an endosomal pathway for cell entry and that proteases like CPL are involved in this mode of entry.

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 The Emergence of Human Coronavirus EMC: How Scared Should We Be?

mBio ◽  
2013 ◽  
Vol 4 (2) ◽  
Author(s):  
Renee W. Y. Chan ◽  
Leo L. M. Poon
Keyword(s):  
Airway Epithelial Cells ◽  
Type I ◽  
Airway Epithelial ◽  
Human Coronavirus ◽  
Angiotensin Converting Enzyme 2 ◽  
Human Airway Epithelium ◽  
Mammalian Cell Lines ◽  
Physiological Relevance

ABSTRACT A novel betacoronavirus, human coronavirus (HCoV-EMC), has recently been detected in humans with severe respiratory disease. Further characterization of HCoV-EMC suggests that this virus is different from severe acute respiratory syndrome coronavirus (SARS-CoV) because it is able to replicate in multiple mammalian cell lines and it does not use angiotensin-converting enzyme 2 as a receptor to achieve infection. Additional research is urgently needed to better understand the pathogenicity and tissue tropism of this virus in humans. In their recent study published in mBio, Kindler et al. shed some light on these important topics (E. Kindler, H. R. Jónsdóttir, M. Muth, O. J. Hamming, R. Hartmann, R. Rodriguez, R. Geffers, R. A. Fouchier, C. Drosten, M. A. Müller, R. Dijkman, and V. Thiel, mBio 4[1]:e00611-12, 2013). These authors report the use of differentiated pseudostratified human primary airway epithelial cells, an in vitro model with high physiological relevance to the human airway epithelium, to characterize the cellular tropism of HCoV-EMC. More importantly, the authors demonstrate the potential use of type I and type III interferons (IFNs) to control viral infection.

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 Differences in neutralizing antigenicity between laboratory and clinical isolates of HCoV-229E isolated in Japan in 2004–2008 depend on the S1 region sequence of the spike protein

2012 ◽  
Vol 93 (9) ◽  
pp. 1908-1917 ◽  
Author(s):  
Kazuya Shirato ◽  
Miyuki Kawase ◽  
Oshi Watanabe ◽  
Chika Hirokawa ◽  
Shutoku Matsuyama ◽  
...  
Keyword(s):  
Protein A ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Pseudotyped Virus ◽  
S Protein ◽  
Human Sera ◽  
Antigenic Phenotype ◽  
Vesicular Stomatitis ◽  
The Common ◽  
The Difference

Human coronavirus (HCoV) is a causative agent of the common cold. Although HCoV is highly prevalent in the world, studies of the genomic and antigenic details of circulating HCoV strains have been limited. In this study, we compared four Japanese isolates with the standard HCoV-229E strain obtained from ATCC (ATCC-VR740) by focusing on the spike (S) protein, a major determinant of neutralizing antigen and pathogenicity. The isolates were found to have nucleotide deletions and a number of sequence differences in the S1 region of the S protein. We compared two of the Japanese isolates with the ATCC-VR740 strain by using virus-neutralizing assays consisting of infectious HCoV-229E particles and vesicular stomatitis virus (VSV)-pseudotyped virus carrying the HCoV-229E S protein. The two clinical isolates (Sendai-H/1121/04 and Niigata/01/08) did not react with antiserum to the ATCC-VR740 strain via the neutralizing test. We then constructed a pseudotype VSV-harboured chimeric S protein with the ATCC S1 and Sendai S2 regions or that with Sendai S1 and ATCC S2 regions and compared them by a neutralization test. The results revealed that the difference in the neutralizing antigenicity depends on the S1 region. This different antigenic phenotype was also confirmed by a neutralizing test with clinically isolated human sera. These results suggest that the HCoV-229E viruses prevalent in Japan are quite different from the laboratory strain ATCC-VR740 in terms of the S sequence and neutralization antigenicity, which is attributed to the difference in the S1 region.

scholarly journals Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells

2012 ◽  
Vol 303 (2) ◽  
pp. L97-L106 ◽  
Author(s):  
Shilpa Nimishakavi ◽  
Marina Besprozvannaya ◽  
Wilfred W. Raymond ◽  
Charles S. Craik ◽  
Dieter C. Gruenert ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Selective Inhibition ◽  
Airway Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Apical Surface ◽  
Airway Epithelial ◽  
Wild Type ◽  
Bronchial Epithelial

Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na+ channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o−) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o− epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.

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.

scholarly journals Estrogen regulates the expression of SARS-CoV-2 receptor ACE2 in differentiated airway epithelial cells

2020 ◽  
Vol 318 (6) ◽  
pp. L1280-L1281 ◽  
Author(s):  
Kimberly E. Stelzig ◽  
Fabrizio Canepa-Escaro ◽  
Marta Schiliro ◽  
Sergejs Berdnikovs ◽  
Y. S. Prakash ◽  
...  
Keyword(s):  
Sexual Dimorphism ◽  
Epithelial Cells ◽  
Severe Acute Respiratory Syndrome ◽  
Angiotensin Converting Enzyme ◽  
Sex Steroids ◽  
Airway Epithelial Cells ◽  
Cell Entry ◽  
Airway Epithelial ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2

There is marked sexual dimorphism in the current coronavirus disease 2019 (COVID-19) pandemic. Here we report that estrogen can regulate the expression of angiotensin-converting enzyme 2 (ACE2), a key component for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry, in differentiated airway epithelial cells. Further studies are required to elucidate the mechanisms by which sex steroids regulate SARS-CoV-2 infectivity.

scholarly journals Intracellular autoactivation of TMPRSS11A, an airway epithelial transmembrane serine protease

2020 ◽  
Vol 295 (36) ◽  
pp. 12686-12696 ◽  
Author(s):  
Ce Zhang ◽  
Yikai Zhang ◽  
Shengnan Zhang ◽  
Zhiting Wang ◽  
Shijin Sun ◽  
...  
Keyword(s):  
Cell Surface ◽  
Serine Proteases ◽  
Transmembrane Domain ◽  
Airway Epithelial Cells ◽  
Surface Proteins ◽  
Site Directed Mutagenesis ◽  
Soluble Form ◽  
Cell Organelle ◽  
Airway Epithelial ◽  
Zymogen Activation

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.

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