Vesicular Stomatitis Virus G Protein Acquires pH-Independent Fusion Activity during Transport in a Polarized Endometrial Cell Line

Growth kinetics of VSV in HEC-1A and HECA2 cell lines. HEC cells were infected with VSV_IND at an MOI of 2. At different times postinfection, released virus was titrated by plaque assay. Viral yields are expressed as PFU per infected cell.

Treatment with NH₄Cl ablates fusion from within. VSV_IND-infected HECA2 cells (MOI = 2) were cultured in the presence of 1 and 20 mM NH₄Cl beginning at 3.5 hpi. At 9 (a, c, and e) and 24 (b, d, and f) hpi, the cells were fixed in methanol and stained with Giemsa stain. Giant cell formation was observed and recorded with an Nikon TMD microscope equipped with a 35-mm camera. (a and b) Untreated VSV_IND-infected HECA2 cells; (c and d) infected HECA2 cells treated with 1 mM NH₄Cl; (e and f) infected HECA2 cells treated with 20 mM NH₄Cl.

Ammonium chloride does not affect viral yields. HECA2 cells were infected with VSV_IND at an MOI of 2 and cultured in the presence or absence of ammonium chloride essentially as described in the legend to Fig. 3. At 24 hpi, the virus released from HECA2 cells was titrated by plaque assay in BHK-21 cells and expressed as PFU per cell.

Treatment with the vacuolar H⁺-ATPase inhibitor Bfm A1 inhibits VSV-induced giant cell formation. VSV_IND-infected HECA2 cells (MOI = 2) were treated with different concentrations of Bfm A1 beginning at 3.5 hpi. At 24 hpi, the cells were fixed in methanol and stained with Giemsa stain. Giant cell formation was completely inhibited at 1, 10, and 100 nM Bfm A1 (a, b, and c, respectively). Giant cell formation could be induced in 10 nM Bfm A1-treated cells following a brief exposure to pH 5.5 buffer (d).

Bfm A1 does not inhibit virus yield. Following a 24-h infection with VSV_IND either in the presence or in the absence of different concentrations of Bfm A1, the virus released from HECA2 cells was titrated by plaque assay in BHK-21 cells. Virus yields are expressed as PFU per cell.

Incorporation of viral proteins into virions in the presence or absence of Bfm A1 and NH₄Cl. BHK-21, HECA2, or HEC-1A cells were infected with VSV_IND at an MOI of 5. Beginning at 3 hpi, the cells were cultured in either 10 nM Bfm A1 or 20 mM NH₄Cl. Infected cells were metabolically labeled with 60 μCi of [³⁵S]Met-Cys per ml from 5 to 20 hpi.³⁵S-labeled virus released into the medium was pelleted by ultracentrifugation and analyzed by SDS-PAGE. Lanes: C, control untreated cells; N, NH₄Cl-treated cells; B, Bfm A1-treated cells.

Cell surface distribution and fusion activity of the viral G protein following Bfm A1 treatment. HECA2, BHK-21, and Vero C1008 cells at subconfluent cell density were infected with VSV_IND at an MOI of 2. Beginning at 3 hpi, the cells were cultured either in the presence (+) or in the absence (−) of 5 nM Bfm A1. At 9 hpi, the cells were briefly exposed to either low pH (pH 5.7) or neutral pH (pH 7.2) as indicated for 1 min at room temperature, subsequently neutralized with incubation medium, and incubated for a further 15 to 20 min at 37°C. Cells were fixed in 3% paraformaldehyde, and viral G protein was detected at the cell surface by using a mouse monoclonal antibody to the viral G protein followed by incubation with an Alexa 488-conjugated goat anti-mouse immunoglobulin secondary antibody. Fluorescence was monitored and photographed with a Nikon Axiophot microscope equipped with epifluorescence and a 35-mm camera.