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P.M. Scanlan, M.–J. Oh, D. Shukla; The Use of a Beta–Galactosidase Alpha–Complementation System to Examine Cell–Cell Fusion Requirements and Fusion Kinetics in Herpes Simplex Virus . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3061.
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The goal of this study was to develop a quantitative and sensitive assay to examine the requirements of cell to cell fusion and fusion kinetics.
Expression of alpha and omega peptides via separate plasmids allowed formation of a functional beta–galactosidase enzyme only when the two peptides found each other. This was detected by color development using x–gal substrate. Virus like cells expressing viral glycoproteins gB, gD, gH, gL, and alpha peptide were mixed with omega peptide and receptor expressing target cells allowing cell fusion to be detected rapidly after cell–cell fusion. This assay was used for determining the rate of fusion, temperature dependence, and the requirements for fusion. The results were compared with standard luciferase–based cell fusion assays.
Alpha–complementation was successful in measuring cell–to–cell fusion showing strong levels of fusion when effector and target cells were mixed. Cell–to–cell fusion occurred within 3 hours post mixing and was receptor dependent. Interestingly, virus like cells that expressed gB, gH, and gL, but not gD showed very low levels of fusion compared to the positive control (5–10%) but significantly higher than background levels in both HSV–1 and HSV–2 expressing cells. Additionally, cell fusion was temperature dependent showing little fusion at 4°C but showed a dramatic increase at temperatures near 24°C.
Alpha–complementation is a useful technique for the study of cell–to–cell fusion yielding easily quantified results, low background, and a short lag time in the reporter enzyme activity (<30min) allowing the study of real time fusion events. This assay showed the detection of fusion at levels just above the background including in the absence of glycoprotein gD. Cell–cell fusion occurs at roughly 3 hours post mixing, and is temperature dependent with cell fusion starting around 24°C.
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