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 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 Receptor-Independent Infection of Murine Coronavirus: Analysis by Spinoculation

2006 ◽  
Vol 80 (10) ◽  
pp. 4901-4908 ◽  
Author(s):  
Rie Watanabe ◽  
Shutoku Matsuyama ◽  
Fumihiro Taguchi
Keyword(s):  
Cell Surface ◽  
Conformational Changes ◽  
Hepatitis Virus ◽  
Quantitative Estimation ◽  
Soluble Form ◽  
Activation Process ◽  
S Protein ◽  
Protein Activation ◽  
Naturally Occurring ◽  
Murine Coronavirus

ABSTRACT A highly neurovirulent murine coronavirus JHMV (wild-type [wt] JHMV) is known to spread from cells infected via the murine coronavirus mouse hepatitis virus receptor (MHVR) to cells without MHVR (MHVR-independent infection), whereas a mutant virus isolated from wt JHMV, srr7, spread only in an MHVR-dependent fashion. These observations were obtained by the overlay of JHMV-infected cells onto receptor-negative cells that are otherwise resistant to wt JHMV infection. MHVR-independent infection is hypothetically thought to be attributed to a naturally occurring fusion activation of the wt JHMV S protein, which did not occur in the case of srr7. Attachment of S protein on cells without MHVR during the S-protein activation process seems to be a key condition. Thus, in the present study, we tried to see whether wt JHMV virions that are attached on MHVR-negative cells are able to infect those cells. In order to make virions attach to the cell surface without MHVR, we have used spinoculation, namely, the centrifugation of cells together with inoculated virus at 3,000 rpm for 2 h. This procedure forces viruses to attach to the cell surface, as revealed by quantitative estimation of attached virions by real-time PCR and also facilitated wt JHMV infection to MHVR-negative cells, but failed to do so for srr7. Virions of both wt and srr7 attached on MHVR-negative cells by spinoculation were facilitated for infection in the presence of a soluble form of MHVR that induces conformational changes of both wt and srr7. It was further revealed that wt JHMV S1, but not srr7, was released from the cell surface when S protein was expressed on cells. These observations support the hypothesis that attachment of the virion to MHVR-negative cells is a critical step and that a unique feature of wt JHMV S1 to be released from S2 in a naturally occurring event is involved in an MHVR-independent 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.

scholarly journals Cyclosporin A inhibits the replication of diverse coronaviruses

2011 ◽  
Vol 92 (11) ◽  
pp. 2542-2548 ◽  
Author(s):  
Adriaan H. de Wilde ◽  
Jessika C. Zevenhoven-Dobbe ◽  
Yvonne van der Meer ◽  
Volker Thiel ◽  
Krishna Narayanan ◽  
...  
Keyword(s):  
Cyclosporin A ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Cyclophilin A ◽  
Strong Inhibition ◽  
Human Coronavirus ◽  
Gfp Reporter ◽  
Rna And Protein Synthesis ◽  
Gfp Reporter Gene ◽  
Human Coronavirus 229E

Low micromolar, non-cytotoxic concentrations of cyclosporin A (CsA) strongly affected the replication of severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus 229E and mouse hepatitis virus in cell culture, as was evident from the strong inhibition of GFP reporter gene expression and a reduction of up to 4 logs in progeny titres. Upon high-multiplicity infection, CsA treatment rendered SARS-CoV RNA and protein synthesis almost undetectable, suggesting an early block in replication. siRNA-mediated knockdown of the expression of the prominent CsA targets cyclophilin A and B did not affect SARS-CoV replication, suggesting either that these specific cyclophilin family members are dispensable or that the reduced expression levels suffice to support replication.

scholarly journals A protocol for adding knowledge to Wikidata: aligning resources on human coronaviruses

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Andra Waagmeester ◽  
Egon L. Willighagen ◽  
Andrew I. Su ◽  
Martina Kutmon ◽  
Jose Emilio Labra Gayo ◽  
...  
Keyword(s):  
Common Ground ◽  
Knowledge Bases ◽  
Public Knowledge ◽  
Medical Problems ◽  
Human Coronavirus ◽  
Data Schema ◽  
Helping Others ◽  
Human Coronaviruses ◽  
The Right ◽  
Human Coronavirus 229E

Abstract Background Pandemics, even more than other medical problems, require swift integration of knowledge. When caused by a new virus, understanding the underlying biology may help finding solutions. In a setting where there are a large number of loosely related projects and initiatives, we need common ground, also known as a “commons.” Wikidata, a public knowledge graph aligned with Wikipedia, is such a commons and uses unique identifiers to link knowledge in other knowledge bases. However, Wikidata may not always have the right schema for the urgent questions. In this paper, we address this problem by showing how a data schema required for the integration can be modeled with entity schemas represented by Shape Expressions. Results As a telling example, we describe the process of aligning resources on the genomes and proteomes of the SARS-CoV-2 virus and related viruses as well as how Shape Expressions can be defined for Wikidata to model the knowledge, helping others studying the SARS-CoV-2 pandemic. How this model can be used to make data between various resources interoperable is demonstrated by integrating data from NCBI (National Center for Biotechnology Information) Taxonomy, NCBI Genes, UniProt, and WikiPathways. Based on that model, a set of automated applications or bots were written for regular updates of these sources in Wikidata and added to a platform for automatically running these updates. Conclusions Although this workflow is developed and applied in the context of the COVID-19 pandemic, to demonstrate its broader applicability it was also applied to other human coronaviruses (MERS, SARS, human coronavirus NL63, human coronavirus 229E, human coronavirus HKU1, human coronavirus OC4).

scholarly journals Examining the persistence of human Coronavirus 229E on fresh produce

2021 ◽  
pp. 103780
Author(s):  
Madeleine Blondin-Brosseau ◽  
Jennifer Harlow ◽  
Tanushka Doctor ◽  
Neda Nasheri
Keyword(s):  
Fresh Produce ◽  
Human Coronavirus ◽  
Human Coronavirus 229E

Effect of pH and temperature on the infectivity of human coronavirus 229E

1989 ◽  
Vol 35 (10) ◽  
pp. 972-974 ◽  
Author(s):  
Alain Lamarre ◽  
Pierre J. Talbot
Keyword(s):  
Incubation Period ◽  
Storage Conditions ◽  
Viral Infectivity ◽  
Human Coronavirus ◽  
Virus Growth ◽  
The Stability ◽  
And Storage ◽  
Influence Of Ph ◽  
Human Coronavirus 229E

The stability of human coronavirus 229E infectivity was maximum at pH 6.0 when incubated at either 4 or 33 °C. However, the influence of pH was more pronounced at 33 °C. Viral infectivity was completely lost after a 14-day incubation period at 22, 33, or 37 °C but remained relatively constant at 4 °C for the same length of time. Finally, the infectious titer did not show any significant reduction when subjected to 25 cycles of thawing and freezing. These studies will contribute to optimize virus growth and storage conditions, which will facilitate the molecular characterization of this important pathogen.Key words: coronavirus, pH, temperature, infectivity, human coronavirus.

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