Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion

Abstract

We have examined mutations in the ectodomain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 within a region immediately adjacent to the membrane-spanning domain for their effect on the outcome of the fusion cascade. Using the recently developed three-color assay (I. Muñoz-Barroso, S. Durell, K. Sakaguchi, E. Appella, and R. Blumenthal, J. Cell Biol. 140:315-323, 1998), we have assessed the ability of the mutant gp41s to transfer lipid and small solutes from susceptible target cells to the gp120-gp41-expressing cells. The results were compared with the syncytium-inducing capabilities of these gp41 mutants. Two mutant proteins were incapable of mediating both dye transfer and syncytium formation. Two mutant proteins mediated dye transfer but were less effective at inducing syncytium formation than was wild-type gp41. The most interesting mutant proteins were those that were not capable of inducing syncytium formation but still mediated dye transfer, indicating that the fusion cascade was blocked beyond the stage of small fusion pore formation. Fusion mediated by the mutant gp41s was inhibited by the peptides DP178 and C34.

FIG. 1

Amino acid sequence and mutations in gp41. FP is the predicted fusion peptide region, and HR1 and HR2 (8) represent, respectively, the N-terminal and C-terminal α-helices of the triple-stranded coiled coil (4, 11). Mutations in the region between HR2 and the TM anchor include deletions of amino acids 665 to 682 and 678 to 682, insertion of a FLAG sequence (YKDDDD), insertion of a DAF sequence (PNKGSGTTS), scrambling of the underlined sequence to SC7 (INNWNFT), and replacement of the five tryptophans with alanines [W(1-5)A]. Peptide C34 represents HR2 amino acids 628 to 663, and peptide DP178 represents amino acids 638 to 673.

FIG. 2

Three-color assay for WT and mutant HIV-1 gp41s. Simian virus 40-based env expression plasmids (1 μg) containing WT (A to D), W(1-5)A (E to H), and +DAF (I to L) env genes were transfected into COS-1 cells in 35-mm plates using DEAE-dextran (1 μg/ml). At 14 h posttransfection, the cells were replated, and starting at 36 to 48 h posttransfection they were incubated with 20 μM CMAC in Dulbecco modified Eagle medium overnight at 37°C. All constructs expressed similar amounts of envelope glycoprotein on the cell surface (8a). The transfected cells were then washed and incubated in fresh medium for 2 h at 37°C before addition of HeLa-CD4 cells which were labeled in the membrane with DiI and in the cytosol with calcein as previously described (7). The COS-1 cells, labeled with CMAC, were cocultured 1:10 at 37°C for 2 h with HeLa-CD4 cells labeled with DiI and calcein, and images were examined by bright-field microscopy (A, E, and I) and fluorescence microscopy for CMAC staining (B, F, and J), for DiI staining (C, G, and K), and for calcein staining (D, H, and L). CMAC is a fluorescent chloromethyl derivative that freely diffuses through the membranes of live cells. Once inside the cell, this mildly thiol-reactive probe undergoes what is believed to be a glutathione S-transferase-mediated reaction to produce membrane-impermeant fluorescent dye adducts with glutathione, as well as with other intracellular components. Staining of COS-1 cells with CMAC gives rise to bright fluorescence due to reaction with proteins in the perinuclear, endoplasmic reticulum, and Golgi regions, which are immobile, as well as to weaker fluorescence due to the fluorescent glutathione adduct (molecular mass, ∼600 Da) in the cytosol, which is able to diffuse through small fusion pores. The COS-1 cells identified by CMAC staining (B, F, and J) are large and often appear multinuclear, although we do not know whether the round granular structures seen by bright-field microscopy of the COS-1 cells are nuclei or large granules. Panels A to D show one large cell triple stained with CMAC, DiI, and calcein (indicated by a star). DiI is internalized after 2 h at 37°C and appears punctate with nuclear sparing due to its localization in membranes of intracellular organelles. Calcein (465 Da) is evenly distributed throughout the cell (D). One large, granular COS-1 cell (A, left) is only stained with CMAC (B); its lack of staining with DiI (C) and calcein (D) indicates that it has not fused with HeLa-CD4 cells. In panel F, a large structure is seen which seems in continuity with CMAC. However, since the bottom left part of this structure is not in continuity with DiI (G) and calcein (H), it represent two cells. The top right cell (indicated by a star) is in continuity with CMAC, DiI, and calcein. Since COS-1 cells expressing W(1-5)A env do not produce blue nuclei when incubated with MAGI cells (see Fig. 3), which requires transfer of the 14-kDa HIV-1 Tat protein (see text), we conclude that this COS-1–HeLa-CD4 conjugate represents a phenotype in which small fusion pores form, allowing movement of lipids and small molecules (<1,000 Da) but not of large molecules. The same phenotype is seen with COS-1 cells expressing DAF env: the COS-1–HeLa-CD4 conjugate indicated by a star in panels J, K, and L is in continuity with CMAC, DiI, and calcein but does not allow transfer of HIV-1 Tat (see Fig. 3).

FIG. 3

Fusogenic activity of WT and mutant HIV-1 gp41s. The three-color assay was performed as described in the legend to Fig. 2. Since multiple rounds of fusion may interfere with quantitation in the case of WT and mutant env genes which produce a large number of blue nuclei after 24 h at 37°C (grey bars), incubations were done for 2 h at 37°C. Black bars represent 100 times the number of COS-1 cells stained with DiI and calcein over the total number of COS-1–HeLa-CD4 conjugates measured in the three-color assay. The data are representative of five separate experiments. In each experiment, a total of 30 to 50 COS-1–HeLa-CD4 conjugates were counted. The number of nuclei per syncytium (grey bars) was obtained from the MAGI assay (8a) and represents the ability of HIV-1 Tat to transfer from COS-1 cells to HeLa-CD4 cells.

FIG. 4

Inhibition of cell-cell fusion by DP178 and C34 peptides. Cell fusion was calculated as a percentage of the control by using the three-color assay method shown in Fig. 2 and 3 and described in the text for the WT, W(1-5)A, and SC7.