A new approach to measure fusion activity of cloned viral envelope proteins: fluorescence dequenching of octadecylrhodamine-labeled plasma membrane vesicles fusing with cells expressing vesicular stomatitis virus glycoprotein.
Fusion between fluorescently labeled plasma membrane vesicles (PMV) and cells expressing vesicular stomatitis virus (VSV) glycoprotein (G-protein) was investigated by utilizing a lipid mixing assay based on fluorescence dequenching of octadecyl rhodamine (R18). The PMVs were prepared from Vero cells by hypotonic lysis. The G-protein was expressed on the cell surface either following infection with intact VSV or with an adenovirus vector (AdG12) containing the gene for the G-protein. Fusion was temperature and pH dependent and was inhibited by VSV G-antiserum. The pH dependence of PMV fusion paralleled that observed for VSV-cell fusion and VSV-induced syncytia formation. The kinetics of fusion followed an exponential dependence on time without an observable time lag after lowering pH. These findings indicate that dequenching R18-labeled PMV reliably represents the basic features of fusion of VSV with cells and can be used as a new tool in the study of fusion activity of virus envelope proteins expressed in cells.
Section of Membrane Structure and Function, NCI, NIH, Bethesda, Maryland 20892.
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Puri
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Krumbiegel
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Dimitrov
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Blumenthal
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008561:Membrane Fusion; D008562:Membrane Glycoproteins; D012235:Rhodamines; D014709:Vero Cells; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism*
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D012235:Rhodamines; D014759:Viral Envelope Proteins; C044542:octadecyl Rhodamine B chloride