Document Detail


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.
MedLine Citation:
PMID:  17553448     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Chih-Fong Chou; Shuo Shen; Geetha Mahadevappa; Seng Gee Lim; Wanjin Hong; Yee-Joo Tan
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2007-04-27
Journal Detail:
Title:  Analytical biochemistry     Volume:  366     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2007 Jul 
Date Detail:
Created Date:  2007-06-18     Completed Date:  2007-08-27     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  190-6     Citation Subset:  IM    
Affiliation:
Institute of Molecular and Cell Biology, Proteos, Singapore 138673. mcbccf@imcb.a-star.edu.sg
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MeSH Terms
Descriptor/Qualifier:
Alkaline Phosphatase / genetics,  metabolism*
Amino Acid Sequence
Animals
Blotting, Western
CHO Cells
COS Cells
Cell Fusion
Cell Line
Cercopithecus aethiops
Cricetinae
Cricetulus
Green Fluorescent Proteins / genetics,  metabolism
Hepacivirus / genetics,  metabolism
Humans
Membrane Fusion*
Membrane Glycoproteins / metabolism*
Models, Biological
Molecular Sequence Data
Peptidyl-Dipeptidase A / metabolism*
Sequence Homology, Amino Acid
Transfection
Viral Envelope Proteins / metabolism*
Viral Nonstructural Proteins / genetics,  metabolism*
Chemical
Reg. No./Substance:
0/Membrane Glycoproteins; 0/Viral Envelope Proteins; 0/Viral Nonstructural Proteins; 107476-75-5/spike glycoprotein, coronavirus; 147336-22-9/Green Fluorescent Proteins; EC 3.1.3.1/Alkaline Phosphatase; EC 3.4.15.1/Peptidyl-Dipeptidase A; EC 3.4.17.-/angiotensin converting enzyme 2

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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