Quantitation of MoMuLV envelope protein on the cell surface.
The envelope glycoprotein (Env) of Moloney murine leukemia virus (MoMuLV) is proteolytically processed and transported to the cell surface where it can be incorporated into budding virions. Cell surface Env is frequently detected using an indirect immunofluorescence assay and fluorescence-activated cell sorting (FACS). We found that the detection of Env in this manner requires the expression of the MoMuLV receptor (ATRC-1) on the cell surface, and the level of envelope protein detected correlates with the level of receptors expressed on the cell. In addition, Env detection corresponds to the Env protein's ability to bind to its receptor and can be competed out by the addition of a truncated form of the Env protein. These data suggest that Env detected on the cell surface by the FACS assay is protein that has rebound to its receptor after being secreted or shed, rather than actual surface-expressed protein. In contrast, a combined immunoprecipitation and biotinylation assay detected equal amounts of Env on the surface of both receptor-lacking and receptor-expressing cell lines. The immunoprecipitation-biotinylation assay is therefore a more appropriate method for detecting surface expression of the MoMuLV envelope protein.
Yu
H
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Gene Therapy Laboratories, Norris Cancer Center, School of Medicine, University of Southern California, Los Angeles 90033, USA.
Empig
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C
Xia
J
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Anderson
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WF
D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011991:Receptors, Virus / Q000378:metabolism*; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism*
D008562:Membrane Glycoproteins; D011991:Receptors, Virus; D015682:Retroviridae Proteins, Oncogenic; D014759:Viral Envelope Proteins; C070506:ecotropic murine leukemia virus receptor