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Chloramphenicol acetyltransferase expression as a sensor for fusion activity.

Membrane fusion is an indispensable biological process that still needs additional study at the molecular level. Fusogenic viral envelope proteins that mediate viral entry into host cells are a useful tool to study the molecular biology of this process, and precise quantification of fusion events is needed for this purpose. A classical method, with limited precision, however, is to monitor formation of polycaryons as a result of cell-cell fusion by light microscopy (11). More sensitive are gene activation assays, and several modifications of this approach have been developed, most of them using β-galactosidase as a reporter (1,2,7,9). As an alternative approach, we describe here a new simplified assay using induction of the chloramphenicol acetyltransferase gene (CAT) as a sensitive indicator of cellular fusion events. In this system, two stably transfected human astrocytoma cell lines are used; both cell lines were derived from U373 MG (ATCC, Rockville, MD, USA). One cell line encodes a chimeric protein of the GAL4 DNA binding and herpes simplex virus (HSV) VP16 activation domains (including a GAL4 nuclear localization sequence) under the control of the simian virus 40 (SV40) early promotor. The other encodes five consensus GAL4 binding sites and the adenoviral E1b promotor upstream of the CAT gene. Fusion between these cell lines leads to transactivation of the reporter gene and is quantified by determination of CAT activity. Cell lines were established following liposome-mediated co-transfection (Life Technologies, Karlsruhe, Germany) of subconfluent U373 MG cells 24 h after seeding (9 cm petri dishes), using 10 μg of constructs pM3-VP16 or pG5CAT (both from CLONTECH Laboratories, Heidelberg, Germany) and 2 μg of pIG-1 (8), a plasmid coding for neomycin resistance. Two days after transfection, selection was initiated with geneticin (G418) at 800 μg/mL. Resistant clones emerging after 2–3 weeks were isolated and, in the case of U373 MG, transfected with the GAL4/ VP16 gene (U373-VP16). Expression was tested by western blotting with specific Polyclonal Antibody (CLONTECH Laboratories). Clones stably transfected with the CAT gene (U373CAT) were identified by cocultivation with U373-VP16 cells followed by polyethylene glycol (PEG)or virusmediated fusion. For this purpose, trypsinized cells were mixed at a ratio of 1:1 and seeded into 35-mm-diameter, 6-well petri plates to obtain a subconfluent monolayer the next day (106 cells). For PEG-mediated fusion, the Benchmarks