The use of hepatitis C virus NS3/4A and secreted alkaline phosphatase to quantitate cell-cell membrane fusion mediated by severe acute respiratory syndrome coronavirus S protein and the receptor angiotensin-converting enzyme 2.
Identifieur interne : 003570 ( Main/Exploration ); précédent : 003569; suivant : 003571The use of hepatitis C virus NS3/4A and secreted alkaline phosphatase to quantitate cell-cell membrane fusion mediated by severe acute respiratory syndrome coronavirus S protein and the receptor angiotensin-converting enzyme 2.
Auteurs : Chih-Fong Chou [Singapour] ; Shuo Shen ; Geetha Mahadevappa ; Seng Gee Lim ; Wanjin Hong ; Yee-Joo TanSource :
- Analytical biochemistry [ 0003-2697 ] ; 2007.
Descripteurs français
- KwdFr :
- Animaux, Cellules CHO, Cellules COS, Cricetinae, Cricetulus, Données de séquences moléculaires, Fusion cellulaire, Fusion membranaire, Glycoprotéine de spicule des coronavirus, Glycoprotéines membranaires (métabolisme), Hepacivirus (génétique), Hepacivirus (métabolisme), Humains, Lignée cellulaire, Modèles biologiques, Peptidyl-Dipeptidase A (métabolisme), Phosphatase alcaline (génétique), Phosphatase alcaline (métabolisme), Protéines de l'enveloppe virale (métabolisme), Protéines virales non structurales (génétique), Protéines virales non structurales (métabolisme), Protéines à fluorescence verte (génétique), Protéines à fluorescence verte (métabolisme), Similitude de séquences d'acides aminés, Séquence d'acides aminés, Technique de Western, Transfection.
- MESH :
- génétique : Hepacivirus, Phosphatase alcaline, Protéines virales non structurales, Protéines à fluorescence verte.
- métabolisme : Glycoprotéines membranaires, Hepacivirus, Peptidyl-Dipeptidase A, Phosphatase alcaline, Protéines de l'enveloppe virale, Protéines virales non structurales, Protéines à fluorescence verte.
- Animaux, Cellules CHO, Cellules COS, Cricetinae, Cricetulus, Données de séquences moléculaires, Fusion cellulaire, Fusion membranaire, Glycoprotéine de spicule des coronavirus, Humains, Lignée cellulaire, Modèles biologiques, Similitude de séquences d'acides aminés, Séquence d'acides aminés, Technique de Western, Transfection.
English descriptors
- KwdEn :
- Alkaline Phosphatase (genetics), Alkaline Phosphatase (metabolism), Amino Acid Sequence, Animals, Blotting, Western, CHO Cells, COS Cells, Cell Fusion, Cell Line, Chlorocebus aethiops, Cricetinae, Cricetulus, Green Fluorescent Proteins (genetics), Green Fluorescent Proteins (metabolism), Hepacivirus (genetics), Hepacivirus (metabolism), Humans, Membrane Fusion, Membrane Glycoproteins (metabolism), Models, Biological, Molecular Sequence Data, Peptidyl-Dipeptidase A (metabolism), Sequence Homology, Amino Acid, Spike Glycoprotein, Coronavirus, Transfection, Viral Envelope Proteins (metabolism), Viral Nonstructural Proteins (genetics), Viral Nonstructural Proteins (metabolism).
- MESH :
- chemical , genetics : Alkaline Phosphatase, Green Fluorescent Proteins, Viral Nonstructural Proteins.
- chemical , metabolism : Alkaline Phosphatase, Green Fluorescent Proteins, Membrane Glycoproteins, Peptidyl-Dipeptidase A, Viral Envelope Proteins, Viral Nonstructural Proteins.
- genetics : Hepacivirus.
- metabolism : Hepacivirus.
- Amino Acid Sequence, Animals, Blotting, Western, CHO Cells, COS Cells, Cell Fusion, Cell Line, Chlorocebus aethiops, Cricetinae, Cricetulus, Humans, Membrane Fusion, Models, Biological, Molecular Sequence Data, Sequence Homology, Amino Acid, Spike Glycoprotein, Coronavirus, Transfection.
Abstract
The membrane fusion process mediated by severe acute respiratory syndrome coronavirus (SARS-CoV) S protein and its cellular receptor angiotensin-converting enzyme 2 (ACE2) had been reconstituted using two Chinese hamster ovary (CHO) cell lines that constitutively express these recombinant proteins separately. This system was applied to develop a quantitative measurement of cell-cell fusion using hepatitis C virus (HCV) NS3/4A protease and a secretion-blocked EGFP-4A/4B-SEAP (EGFP: enhanced green fluorescent protein; 4A/4B: a decapeptide substrate of NS3/4A protease; SEAP: secreted alkaline phosphatase) fusion gene. Both genes were transiently expressed in either of the CHO cell lines, followed by fusion treatment. Significant SEAP activity could be detected in the culture medium only after cell-cell fusion occurred. Cell-cell fusion provides an environment in which the protease encounters its substrate 4A/4B, thereby releasing SEAP from the fusion protein. In this study, we developed a simple, sensitive, and quantitative assay to study the membrane fusion process by measuring the extracellular activity of SEAP.
DOI: 10.1016/j.ab.2007.04.031
PubMed: 17553448
Affiliations:
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Le document en format XML
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scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Cellules CHO</term><term>Cellules COS</term><term>Cricetinae</term><term>Cricetulus</term><term>Données de séquences moléculaires</term><term>Fusion cellulaire</term><term>Fusion membranaire</term><term>Glycoprotéine de spicule des coronavirus</term><term>Humains</term><term>Lignée cellulaire</term><term>Modèles biologiques</term><term>Similitude de séquences d'acides aminés</term><term>Séquence d'acides aminés</term><term>Technique de Western</term><term>Transfection</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The membrane fusion process mediated by severe acute respiratory syndrome coronavirus (SARS-CoV) S protein and its cellular receptor angiotensin-converting enzyme 2 (ACE2) had been reconstituted using two Chinese hamster ovary (CHO) cell lines that constitutively express these recombinant proteins separately. This system was applied to develop a quantitative measurement of cell-cell fusion using hepatitis C virus (HCV) NS3/4A protease and a secretion-blocked EGFP-4A/4B-SEAP (EGFP: enhanced green fluorescent protein; 4A/4B: a decapeptide substrate of NS3/4A protease; SEAP: secreted alkaline phosphatase) fusion gene. Both genes were transiently expressed in either of the CHO cell lines, followed by fusion treatment. Significant SEAP activity could be detected in the culture medium only after cell-cell fusion occurred. Cell-cell fusion provides an environment in which the protease encounters its substrate 4A/4B, thereby releasing SEAP from the fusion protein. In this study, we developed a simple, sensitive, and quantitative assay to study the membrane fusion process by measuring the extracellular activity of SEAP.</div></front></TEI><affiliations><list><country><li>Singapour</li></country></list><tree><noCountry><name sortKey="Hong, Wanjin" sort="Hong, Wanjin" uniqKey="Hong W" first="Wanjin" last="Hong">Wanjin Hong</name><name sortKey="Lim, Seng Gee" sort="Lim, Seng Gee" uniqKey="Lim S" first="Seng Gee" last="Lim">Seng Gee Lim</name><name sortKey="Mahadevappa, Geetha" sort="Mahadevappa, Geetha" uniqKey="Mahadevappa G" first="Geetha" last="Mahadevappa">Geetha Mahadevappa</name><name sortKey="Shen, Shuo" sort="Shen, Shuo" uniqKey="Shen S" first="Shuo" last="Shen">Shuo Shen</name><name sortKey="Tan, Yee Joo" sort="Tan, Yee Joo" uniqKey="Tan Y" first="Yee-Joo" last="Tan">Yee-Joo Tan</name></noCountry><country name="Singapour"><noRegion><name sortKey="Chou, Chih Fong" sort="Chou, Chih Fong" uniqKey="Chou C" first="Chih-Fong" last="Chou">Chih-Fong Chou</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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