Human immunodeficiency virus envelope-dependent cell-cell fusion: a quantitative fluorescence cytometric assay.
BACKGROUND
In vitro fusion of transfected cells expressing the human immunodeficiency virus (HIV) envelope proteins gp120/gp41, with target cells expressing CD4, and a suitable chemokine coreceptor is used widely to investigate the mechanisms of molecular recognition and membrane fusion involved in the entry of the HIV genome into cells and in syncytia formation.


METHODS
We developed an assay that uses two different fluorescent lipophilic probes to single label each reacting cell population and flow cytometry to quantify the extent of cellular fusion after coculture.


RESULTS
Fused cells are detected as double-fluorescent particles in this assay, therefore permitting measurement of their proportion in the total cell population. The time course and extent of HIV-glycoprotein-related cellular fusion, the optimal cell ratio, the size and cell composition of the fusion products, and the inhibition of fusion caused by soluble CD4 and anti-CXCR4 antibody 12G5 were determined. The assay was applied to measure fusion between gp120/gp41 and CD4-expressing cells growing as monolayers (HeLa/CHO fusion), as well as to suspension lymphocyte cultures (Jurkat/Jurkat fusion).


CONCLUSIONS
The method's simple technical and minimal cell-invasive procedures, as well as its non-ambiguous automatic numerical quantification should be useful for the study of factors influencing cell-cell fusion.
Huerta
Leonor
L
Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Distrito Federal, México.
Lamoyi
Edmundo
E
Báez-Saldaña
Armida
A
Larralde
Carlos
C
D000818:Animals; D015704:CD4 Antigens; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004306:Dose-Response Relationship, Immunologic; D005434:Flow Cytometry / Q000379:methods*; D015699:HIV Envelope Protein gp120 / Q000502:physiology*; D015700:HIV Envelope Protein gp41 / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008561:Membrane Fusion / Q000502:physiology*; D015203:Reproducibility of Results; D014162:Transfection
D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41