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Virus-Cell Interactions

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

Paul C. Roberts, Todd Kipperman, Richard W. Compans
Paul C. Roberts
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322
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Todd Kipperman
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322
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Richard W. Compans
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322
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DOI: 10.1128/JVI.73.12.10447-10457.1999
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  • Fig. 1.
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    Fig. 1.

    VSV infection leads to polykaryon formation in HEC cell lines. Cells were infected at an MOI of 2. At 24 hpi, the monolayers were fixed in methanol and stained with Giemsa stain. (a) Mock-infected HEC-1A cells; (b) HEC-1A cells infected with VSVIND; (c and d) mock- and VSVIND-infected HECA2 cells, respectively; (e) HECA2 cells infected with a recombinant vaccinia virus vector encoding the full-length G protein of VSVIND; (f) HECA2 cells infected with VSVNJ/CA.

  • Fig. 2.
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    Fig. 2.

    Growth kinetics of VSV in HEC-1A and HECA2 cell lines. HEC cells were infected with VSVIND 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.

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    Fig. 3.

    Treatment with NH4Cl ablates fusion from within. VSVIND-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 VSVIND-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.

  • Fig. 4.
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    Fig. 4.

    Ammonium chloride does not affect viral yields. HECA2 cells were infected with VSVIND 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.

  • Fig. 5.
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    Fig. 5.

    Treatment with the vacuolar H+-ATPase inhibitor Bfm A1 inhibits VSV-induced giant cell formation. VSVIND-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).

  • Fig. 6.
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    Fig. 6.

    Bfm A1 does not inhibit virus yield. Following a 24-h infection with VSVIND 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.

  • Fig. 7.
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    Fig. 7.

    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 VSVIND 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.

  • Fig. 8.
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    Fig. 8.

    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 VSVIND 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.

  • Fig. 9.
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    Fig. 9.

    VSV-infected HECA2 cells can induce fusion with uninfected target cells. Subconfluent monolayers of HECA2 cells were infected with VSVIND 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. Infected HECA2 cell monolayers were labeled with CellTracker Green CMFDA (green fluorescence) for 40 min at 37°C at 5 hpi. Uninfected target cells, HECA2 (a to c) or BHK-21 (d to f), were labeled with the lipophilic fluorochrome CM-DiI (red-orange fluorescence), trypsinized, and added to the infected cell monolayers at 6.5 hpi. Giant cell formation was monitored from 6 to 12 hpi, at which time monolayers were fixed in 3% paraformaldehyde. Fluorochrome distribution was monitored and photographed with a Nikon Axiophot microscope equipped with epifluorescence and a 35-mm camera. (a and b) HECA2-HECA2 cell fusion at 9 and 12 hpi, respectively (pH 7.2 in the absence of Bfm A1); (c) inhibition of HECA2-HECA2 cell fusion by treatment with Bfm A1 (12 hpi); (d) HECA2-BHK21 cell fusion at 9 hpi (pH 7.2, no Bfm A1); (e and f) HECA2-BHK21 cell fusion at 12 hpi at pH 7.2 and 5.7, respectively. The arrow in panel d points to giant cell formation between infected HECA2 cells.

Tables

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  • Table 1.

    Quantitation of VSV giant cell formation in HECA2 cells

    TreatmentaNo. of S/fieldbNo. of nuclei/synctiumc% Nuclei associated with S
    None
     9 hpi28812.0
     24 hpi34.54888.0
    NH4Cl
     1 mM40.51737.5
     20 mM00
    Bfm A1
     1 nM00
     100 nM00
    Virusd
     VSVIND 365794
     VSVNJ/CA 603083
    • ↵a At 9 hpi, fusion was detected only in untreated cells. All other values were determined at 24 hpi.

    • ↵b The average number of giant cells or syncytia (S) was determined by counting giant cells in random fields of 20× micrographs of DAPI-stained monolayers. Only cells with more than four nuclei per cell were considered syncytia.

    • ↵c Determined by using the formula (mockn − INn)/NS, where mockn is the mean number of single nuclei determined from random fields in noninfected monolayers (mockn = 1,875), INn is the mean number of single nuclei per random 20× field in infected monolayers (data not shown), and NS is the mean number of syncytia per random field.

    • ↵d In a separate experiment, HECA2 cells were mock infected or infected with VSVIND or VSVNJ/CA. Giant cell formation was determined as described above.

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Vesicular Stomatitis Virus G Protein Acquires pH-Independent Fusion Activity during Transport in a Polarized Endometrial Cell Line
Paul C. Roberts, Todd Kipperman, Richard W. Compans
Journal of Virology Dec 1999, 73 (12) 10447-10457; DOI: 10.1128/JVI.73.12.10447-10457.1999

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Vesicular Stomatitis Virus G Protein Acquires pH-Independent Fusion Activity during Transport in a Polarized Endometrial Cell Line
Paul C. Roberts, Todd Kipperman, Richard W. Compans
Journal of Virology Dec 1999, 73 (12) 10447-10457; DOI: 10.1128/JVI.73.12.10447-10457.1999
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KEYWORDS

Macrolides
membrane fusion
Membrane Glycoproteins
Proton-Translocating ATPases
Vacuolar Proton-Translocating ATPases
Vesicular stomatitis Indiana virus
Viral Envelope Proteins

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