A cell-cell fusion assay to assess arenavirus envelope glycoprotein membrane-fusion activity

Joanne York, Jack H. Nunberg

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages157-167
Number of pages11
DOIs
StatePublished - 2018

Publication series

NameMethods in Molecular Biology
Volume1604
ISSN (Print)1064-3745

Keywords

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

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