Netesov. used an assay based on the induction of a -galactosidase (-galactosidase gene (target cells and HeLa cells expressing HIV-1 Tat and either EboV GP or other viral glycoproteins, essentially as explained previously (11, 12). To this end, adherent HeLa cells (2 105 per well; six-well trays) were cotransfected with expression vectors for HIV-1 Tat (1 g) and either EboV GP, HIV-1 Env, HTLV-1 Env, or VSV-G (3 g) by a standard calcium phosphate precipitation technique. Acid treatment, if any, was applied 24 h after transfection by PF-5006739 replacing the culture medium with 3 ml of DMEM-HCl prepared immediately before use at the indicated pH. After 10 min at room heat under a tissue PF-5006739 culture hood, DMEM-HCl was aspirated, and coculture was initiated by adding 4 105 target cells in 2 ml of culture medium. The pH was in the neutral range at the initiation of the coculture. After 24 h, adherent cells were fixed with 0.5% glutaraldehyde in phosphate-buffered saline (PBS) and stained with the -galactosidase substrate X-Gal (5-bromo-4-chloro-3-indolyl-d-galactopyranoside). Blue-stained foci representing fusion events were scored under 4 magnification. Pictures were taken on a Nikon Microphot-FAX microscope. To address the effect of neutralizing antibodies on syncytia formation, anti-EboV IgGs were added at the same time as that of target cells and left during coculture. To investigate the effect of target cell treatment on cell-cell fusion, adherent HeLa-P4 target cells were digested with proteinase K for 1 h before being washed and added to effector cells or incubated 2 h with dextran sulfate and then washed and detached for coculture. For the detection of cell-cell fusion by exchange of cytoplasmic fluorescent dyes, transfected HeLa cells expressing EboV GP or HIV-1 Env were labeled with the green fluorescent probe CTG and target HeLa MMP2 cells with the reddish fluorescent probe CMTMR, as explained previously (1). Briefly, transfected HeLa cells were left in contact with CTG (10 M in PBS) for 45 min at 37C and then washed and returned to standard culture medium for 30 min before low-pH treatment (pH 5). Adherent HeLa cells were labeled by a 30-min contact with CMTMR (8 M in PBS) at 37C, washed, detached with PBS and 1 mM EDTA, and added to adherent CTG-labeled cells for coculture. After 24 h, adherent cells were fixed in situ with 4% formaldehyde PF-5006739 in PBS (10 min) and then washed and examined on a Leica DMIRE2 confocal microscope under 63 magnification. Images were pseudocolored according to their respective emission wavelengths and overlaid using Metamorph software (Universal Imaging Corporation, West Chester, Pa.). Detection of EboV glycoproteins. Expression of wild-type (WT) and mutant EboV GP in HeLa cells was monitored by circulation cytometry 24 h after transient transfection. Cells were stained with the M746/16.4 anti-GP1 MAb (1:1,000 in PBS with 2% fetal calf serum) for 1 h at 4C, followed by FITC-conjugated anti-mouse IgGs (1:50; 45 min at 4C). Cells were then fixed with PBS and 4% formaldehyde and analyzed on an Epics XL circulation cytometer (Coultronics). RESULTS Detection of cell-cell fusion mediated by the Ebola computer virus glycoproteins. The ability of EboV GP to mediate cell-cell fusion was investigated with a content-mixing assay based on the ability of the HIV-1 transactivator Tat to control expression of a stably transfected reporter gene, such as the -galactosidase target cells and cells expressing Tat by a simple -galactosidase assay performed either in cell extracts or in situ by staining fixed cells with a chromogenic substrate, such as X-Gal (blue). Since HeLa cells are permissive to contamination mediated by EboV GP (39), initial attempts to detect cell-cell fusion used the well-characterized HeLa-P4 cell collection (CD4+), in which the LTRtransgene is usually highly responsive to activation by Tat in spite of a particularly low basal level of -galactosidase activity (6). EboV GP (Zaire species) were expressed by transient transfection of HeLa cells, which were found to be relatively resistant to the cytopathic effects of filovirus GP (3, 28) compared to other cell lines tested. The expression vector (a kind gift of M. P. Grange and F. L. Cosset, Lyon, France) was based on an EboV cDNA allowing translation of the full-length (membrane-associated) GP precursor without mRNA editing, which should maximize expression of functional forms of the GP relative to soluble GP. Adherent transfected HeLa cells were exposed to pH 5 medium for 10 min to take into account the expected requirement of EboV GP for activation.