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.
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.
Chou
Chih-Fong
CF
Institute of Molecular and Cell Biology, Proteos, Singapore 138673. mcbccf@imcb.a-star.edu.sg
Shen
Shuo
S
Mahadevappa
Geetha
G
Lim
Seng Gee
SG
Hong
Wanjin
W
Tan
Yee-Joo
YJ
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000085962:Angiotensin-Converting Enzyme 2; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D019556:COS Cells; D002459:Cell Fusion; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D064370:Spike Glycoprotein, Coronavirus; D014162:Transfection; D014759:Viral Envelope Proteins / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; D000085962:Angiotensin-Converting Enzyme 2