Monitoring Viral‐Mediated Membrane Fusion Using Fluorescent Reporter Methods
Open Access
- 1 March 2011
- journal article
- unit
- Published by Wiley in Current Protocols in Cell Biology
- Vol. 50 (1), 26.9.1-26.9.9
- https://doi.org/10.1002/0471143030.cb2609s50
Abstract
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
This publication has 22 references indexed in Scilit:
- Conformational Changes of the HIV-1 Envelope Protein during Membrane Fusion Are Inhibited by the Replacement of Its Membrane-spanning DomainOnline Journal of Public Health Informatics, 2010
- Viral membrane fusionNature Structural & Molecular Biology, 2008
- Common principles and intermediates of viral protein-mediated fusion: the HIV-1 paradigmRetrovirology, 2008
- Structures and Mechanisms of Viral Membrane Fusion Proteins: Multiple Variations on a Common ThemeCritical Reviews in Biochemistry and Molecular Biology, 2008
- A highly sensitive protein-protein interaction assay based on Gaussia luciferaseNature Methods, 2006
- Modification of a viral envelope glycoprotein cell–cell fusion assay by utilizing plasmid encoded bacteriophage RNA polymeraseJournal of Virological Methods, 2005
- Role of the Specific Amino Acid Sequence of the Membrane-Spanning Domain of Human Immunodeficiency Virus Type 1 in Membrane FusionJournal of Virology, 2005
- Progressive Truncations C Terminal to the Membrane-Spanning Domain of Simian Immunodeficiency Virus Env Reduce Fusogenicity and Increase Concentration Dependence of Env for FusionJournal of Virology, 2003
- β-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein–protein interactionsNature Biotechnology, 2002
- Evidence That the Transition of HIV-1 Gp41 into a Six-Helix Bundle, Not the Bundle Configuration, Induces Membrane FusionThe Journal of cell biology, 2000