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. 2002 Dec;76(23):12300-11.
doi: 10.1128/jvi.76.23.12300-12311.2002.

The membrane-proximal domain of vesicular stomatitis virus G protein functions as a membrane fusion potentiator and can induce hemifusion

E Jeetendra  1 , Clinton S Robison, Lorraine M Albritton, Michael A Whitt
Affiliations

The membrane-proximal domain of vesicular stomatitis virus G protein functions as a membrane fusion potentiator and can induce hemifusion

E Jeetendra et al. J Virol. 2002 Dec.

Abstract

Recently we showed that the membrane-proximal stem region of the vesicular stomatitis virus (VSV) G protein ectodomain (G stem [GS]), together with the transmembrane and cytoplasmic domains, was sufficient to mediate efficient VSV budding (C. S. Robison and M. A. Whitt, J. Virol. 74:2239-2246, 2000). Here, we show that GS can also potentiate the membrane fusion activity of heterologous viral fusion proteins when GS is coexpressed with those proteins. For some fusion proteins, there was as much as a 40-fold increase in syncytium formation when GS was coexpressed compared to that seen when the fusion protein was expressed alone. Fusion potentiation by GS was not protein specific, since it occurred with both pH-dependent as well as pH-independent fusion proteins. Using a recombinant vesicular stomatitis virus encoding GS that contained an N-terminal hemagglutinin (HA) tag (GS(HA) virus), we found that the GS(HA) virus bound to cells as well as the wild-type virus did at pH 7.0; however, the GS(HA) virus was noninfectious. Analysis of cells expressing GS(HA) in a three-color membrane fusion assay revealed that GS(HA) could induce lipid mixing but not cytoplasmic mixing, indicating that GS can induce hemifusion. Treatment of GS(HA) virus-bound cells with the membrane-destabilizing drug chlorpromazine rescued the hemifusion block and allowed entry and subsequent replication of GS(HA) virus, demonstrating that GS-mediated hemifusion was a functional intermediate in the membrane fusion pathway. Using a series of truncation mutants, we also determined that only 14 residues of GS, together with the VSV G transmembrane and cytoplasmic tail, were sufficient for fusion potentiation. To our knowledge, this is the first report which shows that a small domain of one viral glycoprotein can promote the fusion activity of other, unrelated viral glycoproteins.

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Figures

FIG. 1.
FIG. 1.
Schematic representation of GS truncations. (A) The amino acid sequence of the membrane-proximal ectodomain of G protein is shown. The numbers at the top of the sequence indicate the positions of the amino acids from the N terminus of G protein and correspond to the residues at which successive truncations of GS were made. The numbers below the sequence denote the number of amino acids from the transmembrane (TM) domain (box). (B) The amino acid sequences of the HA and Flag epitopes that were used to tag the various GS truncations are shown.
FIG. 2.
FIG. 2.
GS enhances membrane fusion induced by GNJ. BHK-21 cells were transfected with plasmids encoding GSHA (A), GSHA and GNJ (B), or GNJ alone (C). At 36 h posttransfection, cells were fixed with 3% paraformaldehyde and stained. The cells were not pretreated with fusion medium, and therefore, the pH was maintained at 7.2 to 7.4 throughout the incubation period. Cells expressing GNJ and GNJ plus GSHA were probed with monoclonal antibody VIII, which is specific for the GNJ glycoprotein. Cells expressing GSHA were stained with the 12CA5 monoclonal antibody, which is specific for the HA epitope. Representative epifluorescence and phase-contrast micrographs of the cells are shown.
FIG. 3.
FIG. 3.
Effect of GS on SV5 F-mediated membrane fusion. BHK-21 cells were transfected with plasmids encoding SV5 F alone or cotransfected with plasmids encoding SV5 F and GSHA. Cells were fixed with 3% paraformaldehyde at 36 h after transfection and stained for surface expression of SV5 F with the monoclonal antibody F1a, which is specific for F protein. (A) Fusion activity of SV5 F alone. (B) Syncytium formation when SV5 F and GSHA were coexpressed.
FIG. 4.
FIG. 4.
GS can function with HIV-1 Env to induce membrane fusion in a chemokine-independent manner. (A) BHK-21 cells were transfected with plasmids encoding either gp160W3IGS alone, CD4 and GSHA together, the chimeric protein CD4-GS alone, or GSHA alone. At 24 h posttransfection, the cells were removed from the plates, divided into two aliquots, and then replated. After an additional 24 h, the HIV-1 Env-expressing cells were stained with the gp160-specific goat serum HT3. Cells expressing CD4 or CD4-GS were stained with the CD4-specific monoclonal antibody Sim.2. Cells expressing GSHA were stained with an HA epitope-specific monoclonal antibody. (B) After 24 h of transfection, the cells expressing CD4 only, GSHA only, CD4 and GSHA together, or CD4-GS were mixed with cells expressing gp160W3IGS and replated. The mixed cultures were incubated for an additional 24 h and were then fixed, processed, and probed with the Sim.2 monoclonal antibody for CD4 expression or for GSHA with monoclonal antibody HA.11.
FIG. 5.
FIG. 5.
Binding of wild-type VSV and GSHA virus to cells. Radiolabeled virions (∼80,000 cpm) were resuspended in binding medium adjusted to pH 7.0 or 5.9. The virus suspensions were added to cells and allowed to bind for 3 h on ice. The inoculum was removed (unbound fraction), and the cells were washed three times with ice-cold binding medium at the same pH to remove unbound virus. Cells were then solubilized with PBS containing 1% Triton X-100, and the lysates were collected (bound fraction). The amount of radioactivity in the bound and unbound fractions was determined by scintillation counting. The binding activity of the viruses is shown as a percentage of the total amount of input virus. Each bar represents an average of three separate experiments done in triplicate. Gray bars, wild-type VSV; solid bars, GSHA virus; hatched bars, ΔG virus.
FIG. 6.
FIG. 6.
Three-color fusion assay. BHK-21 cells were infected with wild-type (wt) VSV (panels A and B), GSHA virus (panel C), or ΔG virus (panel D) at a multiplicity of infection of 10. After 8 h of infection, the cells were labeled with DiI (red) and calcein-AM (green) and then removed from the plates. The cells were then overlaid on BHK-21 cells that had been labeled with CMAC (blue). After the virus-infected donor cells had attached to the plate or settled onto the target cells, the medium was replaced with fusion medium buffered to pH 5.9 or 7.0. After 1 min, the fusion buffer was removed, and the cells were incubated for 20 min in growth medium at 37oC and then placed on ice to prevent any further lipid or content mixing. Phase-contrast and fluorescence images were obtained with filters for 4′,6′-diamidino-2-phenylindole (DAPI) (blue), rhodamine (red), and fluorescein isothiocyanate (FITC) (green). The DAPI filter set did not eliminate bleedthrough of the FITC fluorescence, and therefore the FITC-labeled cells appear greenish-blue in the micrographs. The target cells labeled with CMAC appear as larger, flat blue cells, while the donor cells appear as small, round cells and are labeled both red and green. Relevant target cells are highlighted by a thin white outline in panels A, B, and D. (A) Fusion mediated by wild-type G protein at pH 5.9. The phase-contrast image shows one small donor cell adjacent to two target cells. Transfer of both DiI and calcein-AM from the donor cell to the target cell (blue) can be seen (arrows). (B) A second plate identical to the one shown in panel A but maintained at pH 7.0 shows a lack of transfer of DiI or calcein-AM to the outlined target cell, consistent with the requirement for low pH in initiating VSV G-mediated membrane fusion. (C) Fusion mediated by GSHA at pH 7.0. Several donor cells in contact with target cells are shown. Only the transfer of DiI from the donor to the target cells was seen (arrows). There was no transfer of calcein-AM, showing that complete fusion had not occurred. (D) Multiple donor cells infected with ΔG-VSV which are in contact with an outlined blue target cell are shown, but no transfer of either DiI or calcein-AM occurred.
FIG. 7.
FIG. 7.
Chlorpromazine rescues GSHA virus infectivity. Equivalent amounts of GSHA-GFP virus, ΔG (DelG)-GFP virus, and VSV-GFP virus were resuspended in binding medium buffered to pH 7.0 and incubated at room temperature for 30 min. Following the incubation, the virus suspension was cooled on ice for 10 min and then added to BHK-21 cells on ice. Following 3 h of binding on ice, the inoculum was removed, and the cells were washed with fresh medium to remove the unbound virus. Cells were then treated with medium containing 0.4 mM CPZ for 1 min. The drug-containing medium was removed immediately, and the cells were incubated in drug-free medium overnight. Twelve hours later, the number of cells expressing GFP was counted. The open boxes show the number of GFP-positive cells in the absence of CPZ, while the hatched boxes indicate the number of GFP-positive cells after CPZ treatment. VSV-GFP infected more than 90% of the cells in the presence or absence of CPZ, and the GFP cell count is not shown.
FIG. 8.
FIG. 8.
Fusion potentiation by GS deletion mutants. BHK-21 cells were cotransfected with plasmids encoding SV5 F and either GFP or the GS truncation mutants that were tagged with the Flag epitope. At 36 h posttransfection, the cells were fixed and probed with the F1a monoclonal antibody to detect SV5 F protein. Rhodamine-labeled goat anti-mouse immunoglobulin antibody was used to visualize the cells. Phase-contrast and epifluorescence images were obtained with a 10× water immersion lens with a Zeiss Axiophot microscope. At least 10 images were obtained for each sample. Representative photomicrographs of cells expressing GS with either 14 amino acids (N449) or 9 amino acids (V454) are shown.
FIG. 9.
FIG. 9.
Model for fusion potentiation of heterologous viral glycoproteins by GS. The membrane at the top of each step represents the target membrane, while the membrane at the bottom, which contains the fusion protein (depicted as a trimeric molecule with globular heads) and GS (depicted as trimeric rectangles), represents the donor membrane. Panels A to E illustrate the pathway towards complete fusion between the two membranes mediated by the viral fusion protein and GS. (A) Two individual membranes before the initiation of fusion. The microdomains where GS is located are shown as having increased membrane curvature. (B) GS alone or GS and the fusion protein bind to the target membrane, which results in close membrane apposition and the initiation of conformational changes in the fusion protein. In pathway I, the fusion protein undergoes additional conformational changes that result in exposure of the fusion peptide, which immediately inserts into the target membrane, as shown in panel C. (D) The glycoprotein undergoes further conformational changes that result in formation of the “hairpin” loop intermediate, which is the driving force for membrane merger. Destabilization of the target membrane by the interaction of GS as well as changes induced by the fusion protein cause the formation of a hemifusion diaphragm. In pathway II, the binding events are sufficient to cause GS-mediated hemifusion diaphragm formation (D), which then triggers activation of the fusion protein, release of the fusion peptide, and formation of a hemifusion diaphragm. The unequal forces exerted on the membranes by the lateral movement of protein molecules result in the formation of a fusion pore (E), which then enlarges and completes the fusion reaction (F).
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