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A Cell-Cell Fusion Assay to Assess Arenavirus Envelope Glycoprotein Membrane-Fusion Activity

Part of the Methods in Molecular Biology book series (MIMB,volume 1604)

Abstract

For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a β-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the β-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.

Key words

  • Arenavirus
  • Envelope glycoprotein
  • Membrane fusion
  • Syncytium formation
  • Cell-cell fusion
  • Endosome
  • Fusion inhibitor

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Acknowledgments

Our studies of arenavirus membrane fusion and its inhibition have been funded over the past decade by the National Institutes of Health through the following research grants: R21 AI059355, U54 AI065357 (Rocky Mountain Center for Excellence in Biodefense and Emerging Infectious Diseases; John Belisle, Colorado State University), R01 AI074818, R01 AI093387 (Partnerships for Biodefense; Sean Amberg, SIGA Technologies), and R21 AI120490.

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Correspondence to Jack H. Nunberg .

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York, J., Nunberg, J.H. (2018). A Cell-Cell Fusion Assay to Assess Arenavirus Envelope Glycoprotein Membrane-Fusion Activity. In: Salvato, M. (eds) Hemorrhagic Fever Viruses. Methods in Molecular Biology, vol 1604. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6981-4_10

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  • DOI: https://doi.org/10.1007/978-1-4939-6981-4_10

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  • Publisher Name: Humana Press, New York, NY

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