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Method for Measurement of Viral Fusion Kinetics at the Single Particle Level
Published on: September 7, 2009
Studies of HIV-1 envelope glycoprotein-mediated fusion using a simple fluorescence assay
C D Weiss1, S W Barnett, N Cacalano
1Department of Pharmacology, University of California, San Francisco, USA.
Insights
HIV envelope glycoprotein (Env) mediates cell-cell fusion, a slow process requiring gp41. This study used a sensitive assay to show fusion occurs even without syncytia formation, highlighting Env
Area of Science:
- Virology
- Cell Biology
- Immunology
Background:
- HIV entry into host cells is primarily mediated by the viral envelope glycoprotein (Env).
- Understanding the mechanisms of Env-mediated fusion is crucial for developing antiviral strategies.
Purpose of the Study:
- To investigate the kinetics and characteristics of HIV envelope glycoprotein (Env)-mediated cell-cell fusion.
- To utilize a sensitive fusion assay to monitor Env-mediated entry processes.
Main Methods:
- Utilized fluorescently labeled CD4+ lymphocytes or T-cell lines.
- Monitored fusion by observing dye transfer between labeled CD4+ cells and Env-expressing adherent cells.
- Investigated fusion kinetics at 37°C and assessed the role of pre-binding at 4°C.
Main Results:
- Cell-cell fusion initiated within 20-30 minutes at 37°C, with no significant lag reduction from pre-binding.
- Non-syncytium-inducing (NSI) HIV Env strains mediated dye transfer but not syncytia formation.
- Glycosylphosphatidylinositol (GPI)-anchored Env was incapable of mediating membrane fusion; specific cell types did not support fusion.
Conclusions:
- HIV Env-mediated cell-cell fusion is a gradual, multi-step process dependent on the gp41 transmembrane domain after CD4 binding.
- Fusion can occur between individual cells even in the absence of syncytia formation, challenging assumptions based solely on syncytia phenotype.
- The findings provide insights into the complex mechanisms of HIV entry and cell fusion.
Objective:
To study HIV envelope glycoprotein (Env)-mediated entry using a sensitive fusion assay.
Design And Methods:
CD4+ lymphocytes or T-cell lines were labelled with fluorescent cytoplasm or membrane markers. Fusion with Env-expressing adherent cells was monitored by observing dye transfer from CD4+ cells to Env cells.
Results:
Cell-cell fusion began 20-30 min after co-cultivation at 37 degrees C. Pre-binding at 4 degrees C was observed not to decrease the lag phase before fusion. Cells expressing envelope glycoproteins from non-syncytium-inducing (NSI) HIV strains showed dye transfer between two cells without progression to syncytia. A glycosylphosphatidylinositol anchored Env was found to be incapable of mediating membrane fusion, as measured either by lipid or cytoplasm contents mixing. Primary mouse cells expressing human CD4 and mouse 3T3 cells stably expressing both human CD4 and human CD26 did not support fusion with our Env-expressing cells.
Conclusions:
Env-mediated cell-cell fusion is a relatively slow process, probably reflecting a multi-step process occurring after CD4 binding and requiring the transmembrane domain of gp41. Env proteins are able to mediate cell-cell fusion at least under some experimental conditions, indicating that lack of a syncytia phenotype does not rule out the possibility of fusion occurring between only two or a few cells.
