Monitoring Viral‐Mediated Membrane Fusion Using Fluorescent Reporter Methods

Naoyuki Kondo1, Kosuke Miyauchi2, Zene Matsuda1

1 China‐Japan Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China, 2 AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 26.9
DOI:  10.1002/0471143030.cb2609s50
Online Posting Date:  March, 2011
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A simple and real‐time cell‐based assay of membrane fusion employing a pair of engineered novel reporter proteins is described. The reporter proteins are chimeras of split Renilla luciferase (RL) and split green fluorescent protein (GFP). This reporter allows us to perform both quantitative (RL mode) and visible (GFP mode) membrane fusion assays in live cells. The kinetic assay enabled by this method helps understand the mechanism of membrane fusion mediated by a viral envelope protein. This assay system is also suitable for the screening of potential inhibitors. The timing of inhibition by a particular inhibitor can be analyzed by time‐dependent addition of the inhibitor. Although this unit demonstrates the application of the method for the analysis of HIV‐1 envelope protein, the reporter can be applied to analyses of various other viral envelope proteins. Curr. Protoc. Cell Biol. 50:26.9.1‐26.9.9. © 2011 by John Wiley & Sons, Inc.

Keywords: membrane fusion; viral envelope protein; Renilla luciferase; GFP; split protein; HIV‐1; real‐time assay

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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Membrane Fusion in Cell‐to‐Cell Fusion System Using Dual Functional Reporters
  • Alternate Protocol 1: Application of DSP Assay
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Analysis of Membrane Fusion in Cell‐to‐Cell Fusion System Using Dual Functional Reporters

  • 293FT cells (Invitrogen)
  • 293CD4 cells (see Miyauchi et al., )
  • Complete medium: Dulbecco's modified eagle medium (DMEM; Sigma) containing 10% fetal bovine serum (FBS; Hyclone)
  • pNHcRedEluc: Env expression vector (Wang et al., )
  • pDSP 1‐7: Dual split protein 1‐7 expression vector (Kondo et al., )
  • pDSP 8‐11: Dual split protein 8‐11 expression vector (Kondo et al., )
  • Fugene HD (Roche)
  • EnduRen (Promega)
  • Dual‐Glo luciferase reagent (Promega)
  • Packard View Plate (with black wall; Perkin Elmer)
  • 6‐cm UpCell dish (Nunc)
  • 37°C, 5% CO 2 incubator
  • Glomax, luminometer (Promega)
  • Fluorescent microscope (Olympus)

Alternate Protocol 1: Application of DSP Assay

  • Soluble CD4 (R&D Systems)
  • C34 inhibitory peptide (SciLight Biotechnology)
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Literature Cited

Literature Cited
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