Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Dec;76(23):12123-34.
doi: 10.1128/jvi.76.23.12123-12134.2002.

Antigenic properties of the human immunodeficiency virus transmembrane glycoprotein during cell-cell fusion

Catherine M Finnegan  1 , Werner Berg, George K Lewis, Anthony L DeVico
Affiliations

Antigenic properties of the human immunodeficiency virus transmembrane glycoprotein during cell-cell fusion

Catherine M Finnegan et al. J Virol. 2002 Dec.

Abstract

Human immunodeficiency virus (HIV) entry is triggered by interactions between a pair of heptad repeats in the gp41 ectodomain, which convert a prehairpin gp41 trimer into a fusogenic three-hairpin bundle. Here we examined the disposition and antigenic nature of these structures during the HIV-mediated fusion of HeLa cells expressing either HIV(HXB2) envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various lengths of time and then arrested. Fusion intermediates were then examined for reactivity with various monoclonal antibodies (MAbs) against immunogenic cluster I and cluster II epitopes in the gp41 ectodomain. All of these MAbs produced similar staining patterns indicative of reactivity with prehairpin gp41 intermediates or related structures. MAb staining was seen on Env cells only upon exposure to soluble CD4, CD4-positive, coreceptor-negative cells, or stromal cell-derived factor-treated target cells. In the fusion system, the MAbs reacted with the interfaces of attached Env and target cells within 10 min of coculture. MAb reactivity colocalized with the formation of gp120-CD4-coreceptor tricomplexes after longer periods of coculture, although reactivity was absent on cells exhibiting cytoplasmic dye transfer. Notably, the MAbs were unable to inhibit fusion even when allowed to react with soluble-CD4-triggered or temperature-arrested antigens prior to initiation of the fusion process. In comparison, a broadly neutralizing antibody, 2F5, which recognizes gp41 antigens in the HIV envelope spike, was immunoreactive with free Env cells and Env-target cell clusters but not with fused cells. Notably, exposure of the 2F5 epitope required temperature-dependent elements of the HIV envelope structure, as MAb binding occurred only above 19 degrees C. Overall, these results demonstrate that immunogenic epitopes, both neutralizing and nonneutralizing, are accessible on gp41 antigens prior to membrane fusion. The 2F5 epitope appears to depend on temperature-dependent elements on prefusion antigens, whereas cluster I and cluster II epitopes are displayed by transient gp41 structures. Such findings have important implications for HIV vaccine approaches based on gp41 intermediates.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Anti-gp41 MAbs against cluster I and cluster II epitopes react with transient gp41 structures during cell-cell fusion. CellTracker Green-labeled Env cells were cocultivated with HeLa/CD4 target cells for the indicated times as described in Materials and Methods. Immunostaining was performed with BS3-fixed cells (shown) or with unfixed cells cooled to 4°C. Results are shown with the anti-cluster I MAb 50-69 (1.5 μg/ml) and the anti-cluster II MAb 98-6 (1 μg/ml), used at concentrations that produced the optimum signals. Other anti-cluster I MAbs, F240, 240-D, 3D6, and 246-D, produced identical results (data not shown), as did the anti-cluster II MAb 126-6 (data not shown). Cell nuclei appear as gray areas in stained syncytia. Fusion of Env and target cells produces the light green cytoplasmic staining surrounding the nuclei. All antibodies were tested in parallel along with human isotype control immunoglobulin, which produced no binding signal (data not shown). Images representative of at least three experiments are shown. Bars, 10 μm.
FIG. 2.
FIG. 2.
MAbs 50-69 and 98-6 bind to HIV envelope-expressing cells after treatment with sCD4. (A) Env cells were either left untreated or incubated with 2 μg of sCD4/ml in DMEM for 60 min at 4°C and were subsequently stained with MAb 50-69 (1.5 μg/ml) or 98-6 (1 μg/ml). Antibody staining was detected by PE-conjugated goat anti-human IgG. Human isotype control immunoglobulin was tested with treated cells (shown as an overlay on each histogram) for comparison. Control experiments repeated with untreated cells produced the same results as with treated cells. (B) Env cells were incubated with 2 μg of sCD4/ml in DMEM for 60 min at 4°C and subsequently double stained with the gp41 MAb 50-69 (1.5 μg/ml) or 98-6 (1 μg/ml) and an Alexa 594-labeled 17b Fab fragment (red). gp41 MAb staining was detected by an Alexa 488-conjugated goat anti-human secondary antibody (green). Superimposition of gp41 and 17b images produces a yellow stain indicating colocalized antibody binding. All antibodies were tested in parallel along with human isotype control immunoglobulin, which produced no detectable binding signal (data not shown). Images representative of at least three experiments are shown. Bar, 10 μm.
FIG. 3.
FIG. 3.
gp41 structures that express cluster I and cluster II epitopes colocalize with gp120-CD4-coreceptor tricomplexes at contact interfaces during cell-cell fusion. CellTracker Blue-labeled Env cells were cocultivated with target cells for the indicated times as described in Materials and Methods. The cocultured cells were then fixed with BS3 and treated with the anti-cluster I MAb 50-69 (1. 5 μg/ml), the anti-cluster II MAb 98-6 (1 μg/ml), the anti-gp120 MAb M77 (5 μg/ml), and the anti-gp120-CD4-coreceptor tricomplex MAb 8F101 (5 μg/ml) as described in Materials and Methods. All MAbs were used at concentrations that produced optimum binding signals. Anti-gp41 MAb staining appears as a red signal; M77 or 8F101 binding appears as a green signal. Cell nuclei appear as gray areas in the stained syncytia. Fusion of Env and target cells produces the light blue cytoplasmic staining surrounding the nuclei. Arrows indicate the colocalization of MAbs at various time points. All antibodies were tested in parallel along with human isotype control immunoglobulin, which produced no binding signal (data not shown). Representative images are shown. Each experiment was repeated at least three times with the same results. Bars, 10 μm.
FIG. 4.
FIG. 4.
Exposure of cluster I and cluster II epitopes during cell-cell fusion is induced by gp120-CD4 complex formation. (A and B) CellTracker Green-labeled Env cells and target cells were pretreated with 5 μg of SDF-1/ml for 1 h at 37°C prior to cocultivation. The cells were then incubated for 120 min as described in Materials and Methods in the presence of 5 μg of SDF-1/ml. Interacting cells were stained with the indicated MAbs at 1.5 μg/ml (MAb 50-69) and 1 μg/ml (MAb 98-6). (C and D) CellTracker Green-labeled Env cells were cocultivated for 120 min with U373/CD4 target cells, which express CD4 in the absence of a coreceptor, as described in Materials and Methods. Interacting cells were stained with the indicated MAbs at 1.5 μg/ml (MAb 50-69) and 1 μg/ml (MAb 98-6). All antibodies were tested in parallel along with human isotype control immunoglobulin, which produced no binding signal (data not shown). Representative images are shown. Each experiment was repeated at least three times with the same results. Bar, 10 μm.
FIG. 5.
FIG. 5.
MAbs against cluster I and cluster II epitopes do not inhibit cell-cell fusion. (A) Env and target cells were cocultivated at 37°C for 10 min as described in Materials and Methods. Unbound target cells were removed by washing, and the remaining attached Env and target cells were temperature arrested at 23°C. The arrested cells were then incubated for 120 min at 23°C with MAb 240-D (55 nM), 246-D (79 nM), 50-69 (266 nM), 98-6 (66 nM), or 126-6 (84 nM). Normal human IgG (66 nM) and T20 (66 nM) were tested as controls. The treated cells were then returned to a fusogenic temperature (37°C) and cocultured for 15 to 18 h. (B) Env cells were incubated with 2 μg of sCD4/ml in DMEM for 60 min at 4°C. The treated Env cells were washed to remove unbound sCD4 and then incubated for 60 min at 37°C with the same MAbs and controls as for panel A. Target cells were then mixed with Env cells and cocultivated at 37°C. In both assay formats, cell-cell infection was determined after incubation for 18 h at 37°C by a quantitative β-galactosidase assay. Percent inhibition of fusion for each test condition was calculated relative to the level of fusion observed in control assays carried out in the absence of antibodies. Averages from triplicate assays are shown. The results shown are from a representative experiment repeated several times with the same results.
FIG. 6.
FIG. 6.
MAb 2F5 demonstrates temperature-dependent binding to Env cells. (A) CellTracker Green-labeled Env cells were cocultivated with target cells for the indicated times as described in Materials and Methods. Immunostaining was performed as described with MAb 2F5 (15 μg/ml) on BS3-fixed cells at 37°C. MAb 2F5 binding was tested in parallel along with a human isotype control immunoglobulin, which produced no binding signal (data not shown). Representative images from at least three separate experiments are shown. Bars, 10 μm. (B) Env cells were incubated with MAb 2F5 (15 μg/ml) for 30 min at various temperatures as described in Materials and Methods. Cells were then fixed, and MAb binding was detected with an Alexa 594-conjugated secondary antibody (red). MAb binding was tested in parallel along with a human isotype control immunoglobulin, which produced no binding signal (data not shown). Images representative of at least three experiments are shown. Bar, 10 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, P. M. Murphy, and E. A. Berger. 1996. CC CKR5: a RANTES, MIP-1α, MIP-1β receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955-1958. - PubMed
    1. Baker, K. A., R. E. Dutch, R. A. Lamb, and T. S. Jardetzky. 1999. Structural basis for paramyxovirus-mediated membrane fusion. Mol. Cell 3:309-319. - PubMed
    1. Buchacher, A., R. Predl, K. Strutzenberger, W. Steinfellner, A. Trkola, M. Purtscher, G. Gruber, C. Tauer, F. Steindl, A. Jungbauer, et al. 1994. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res. Hum. Retrovir. 10:359-369. - PubMed
    1. Bullough, P. A., F. M. Hughson, J. J. Skehel, and D. C. Wiley. 1994. Structure of influenza haemagglutinin at the pH of membrane fusion. Nature 371:37-43. - PubMed
    1. Cavacini, L. A., C. L. Emes, A. V. Wisnewski, J. Power, G. Lewis, D. Montefiori, and M. R. Posner. 1998. Functional and molecular characterization of human monoclonal antibody reactive with the immunodominant region of HIV type 1 glycoprotein 41. AIDS Res. Hum. Retrovir. 14:1271-1280. - PubMed

Publication types