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. 2004 Jan;78(2):811-20.
doi: 10.1128/jvi.78.2.811-820.2004.

Role of the ectodomain of the gp41 transmembrane envelope protein of human immunodeficiency virus type 1 in late steps of the membrane fusion process

Séverine Bär  1 , Marc Alizon
Affiliations

Role of the ectodomain of the gp41 transmembrane envelope protein of human immunodeficiency virus type 1 in late steps of the membrane fusion process

Séverine Bär et al. J Virol. 2004 Jan.

Abstract

The membrane fusion process mediated by the gp41 transmembrane envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) was addressed by a flow cytometry assay detecting exchanges of fluorescent membrane probes (DiI and DiO) between cells expressing the HIV-1 envelope proteins (Env) and target cells. Double-fluorescent cells were detected when target cells expressed the type of chemokine receptor, CXCR4 or CCR5, matching the type of gp120 surface envelope protein, X4 or R5, respectively. Background levels of double-fluorescent cells were observed when the gp120-receptor interaction was blocked by AMD3100, a CXCR4 antagonist. The L568A mutation in the N-terminal heptad repeat (HR1) of gp41 resulted in parallel inhibition of the formation of syncytia and double-fluorescent cells, indicating that gp41 had a direct role in the exchange of fluorescent probes. In contrast, three mutations in the loop region of the gp41 ectodomain, located on either side of the Cys-(X)(5)-Cys motif (W596 M and W610A) or at the distal end of HR1 (D589L), had limited or no apparent effect on membrane lipid mixing between Env(+) and target cells, while they blocked formation of syncytia and markedly reduced the exchanges of cytoplasmic fluorescent probes. The loop region could therefore have a direct or indirect role in events occurring after the merging of membranes, such as the formation or dilation of fusion pores. Two types of inhibitors of HIV-1 entry, the gp41-derived peptide T20 and the betulinic acid derivative RPR103611, had limited effects on membrane exchanges at concentrations blocking or markedly reducing syncytium formation. This finding confirmed that T20 can inhibit the late steps of membrane fusion (post-lipid mixing) and brought forth an indirect argument for the role of the gp41 loop region in these steps, as mutations conferring resistance to RPR103611V were mapped in this region (I595S or L602H).

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Figures

FIG. 1.
FIG. 1.
Detection of membrane exchanges mediated by the HIV-1 envelope proteins (Env) between cells labeled with the fluorescent lipophilic probe DiI (red) or DiO (green). (A to C) Two-color flow cytometry analysis of HeLa-P4 cells (CD4+ and CXCR4+) labeled with DiI (A), of HeLa-Env/LAI cells labeled with DiO (B), and of a 2-h coculture of these two cell types (C) was initiated by adding HeLa-P4 cells to an equivalent number of adherent HeLa-Env/LAI cells. In each panel, ∼5,000 fluorescent cells were analyzed. Quadrants 1 to 4 were defined for DiI-labeled cells so that >98% wereincluded in quadrant 1 (A). Since a fraction of the DiO-labeled cells had a detectable level of red fluorescence (quadrant 2 in panel B), only cells with the higher red fluorescence corresponding to subquadrant 2a in panel C were scored as double fluorescent. (D) Mean fluorescence intensity (MFI) at the DiI wavelength for DiO-labeled HeLa-Env/LAI cells (lane 1) and for cocultures of DiI-labeled HeLa-P4 cells with DiO-labeled HeLa-Env/LAI (lane 2), HeLa-Env/ADA (lane 3), or Env-negative HeLa cells (lane 4). Cocultures were performed and analyzed as described for panel C. The MFI at the DiI wavelength was calculated for the DiO-positive cells (corresponding to quadrants 2 and 4). Bars are the mean values from three independent experiments.
FIG. 2.
FIG. 2.
Chemokine receptor and gp120 requirements for membrane exchanges and syncytium formation. Quantification of syncytia formed after overnight coculture (A) and of double-fluorescent cells formed after 2-h coculture of HeLa-P4 cells or U373-CCR5 target cells with an equivalent number of Env-negative HeLa cells (lanes 1 and 4), X4 Env+ cells (HeLa-Env/LAI, lanes 2 and 5) or R5 Env+ cells (HeLa-Env/ADA, lanes 3 and 6) (B). Cocultures were initiated by adding ∼2 × 105 target cells to an equivalent number of adherent cells. Target cells were stably transfected with a Tat-inducible lacZ transgene, and their coculture partners expressed HIV-1 Tat, which allowed us to detect syncytia as blue foci after staining with X-Gal. In panel B, target cells were labeled with DiI, and coculture partners were labeled with DiO. Approximately 5,000 cells were analyzed by flow cytometry, and the percentage of double-fluorescent cells was determined as described in the legend of Fig. 1. Results represent the mean values from three independent experiments ± standard errors of the means.
FIG. 3.
FIG. 3.
Effect of inhibitors of HIV-1 entry on membrane exchanges and syncytium formation. Cocultures of DiI-labeled HeLa-P4 cells and DiO-labeled HeLa-Env/LAI cells were initiated at 4°C in the absence of inhibitor (2 h) and then shifted to 37°C in the presence or absence of the indicated concentration of AMD3100 bicyclam (A), betulinic acid derivative RPR103611 (B), or T20 peptide (C). Double-fluorescent cells were quantified by flow cytometry after 3 h at 37°C as described in the legend of Fig. 1, while syncytia were scored the next day in parallel cocultures by staining with X-Gal. The inhibition of membrane exchanges was calculated after subtracting the background level of double-fluorescent cells corresponding to coculture of labeled HeLa-P4 and Env-negative HeLa cells. Results are the mean values from two (T20) or three independent experiments.
FIG. 4.
FIG. 4.
Position of mutations in the HIV-1 gp41 ectodomain. (A) Schematic organization of retroviral transmembrane envelope glycoproteins. FP, fusion peptide; HR1 and HR2, N- and C-terminal helix regions; C—C, conserved Cys residues with disulfide link; MSD, membrane-spanning domain. (B) Partial sequence of the gp41 of HIV-1 LAI and of the homologous region from SIVagm. The positions of mutations are shown with the type of amino acid substituted. Numbering corresponds to the HIV-1 Env precursor (initial residue of gp41 at position 511).
FIG. 5.
FIG. 5.
Effect of mutations in gp41 on membrane exchanges and syncytium formation. (A) Expression of WT and mutant HIV-1 Env determined by flow cytometry after staining transfected HEK293T cells with a polyclonal anti-gp120 antibody and FITC-coupled secondary antibody. Bars represent mean channel fluorescence (arbitrary log units). (B) Formation of syncytia after overnight cocultures of ∼2 × 105 HeLa-P4 cells and an equivalent number of transfected HEK293T cells. Syncytia were scored as blue foci after staining with X-Gal, or they were scored as multinucleated structures in the case of mutant W596M, due to the induction of a high β-galactosidase background upon transfection. (C) Formation of double-fluorescent cells after 2-h coculture of DiI-labeled HeLa-P4 cells and DiO-labeled transfected HEK293T cells. Coculture and flow cytometry analysis were performed as described in the legend of Fig. 1. All the gp41 mutants were tested in parallel. Results represent the means of three or four experiments ± standard errors of the means.
FIG. 6.
FIG. 6.
Detection of membrane exchanges by confocal microscopy. Cocultures were initiated by adding DiI-labeled HeLa-P4 cells onto adherent DiO-labeled HeLa cells (glass coverslips) that were either Env negative (a) or stably expressing WT LAI Env (b) or the derived mutant Env D589L (c) or W596M (d). Cells were fixed after 2 h and analyzed on a Leica DMIRE2 microscope under ×63 magnification, ×2.5 zoom. Images were pseudocolored according to their respective emission wavelengths and overlaid by using Metamorph software.
FIG. 7.
FIG. 7.
Detection of cytoplasmic exchanges mediated by WT or mutant HIV-1 Env with different fluorescent probes. (A) HEK293T cells were transfected to express WT or mutant HIV-1 Env and the GFP and left in contact with HeLa-P4 cells labeled with the cytoplasmic dye CMTMR (red fluorescence). After a 2-h coculture, cells were detached, fixed, and analyzed by two-color flow cytometry. Bars represent percentages of cells with emission that was >3 log units in the red and green wavelengths (means of two experiments). The GFP and Env vectors were in a 1/4 ratio. A control plasmid was used instead of the Env vector to determine background. (B) Results of an experiment identical to that described in panel A, except that HEK293T cells were transfected only with Env vectors and then labeled by uptake of a low-molecular-weight green fluorescent probe, CTG. Cells were fixed in situ after coculture and observed under fluorescence microscopy. Bars represent the numbers of cells emitting fluorescence in both the green and red wavelengths counted in three random fields at ×40 magnification (results are the means ± standard errors of the means of two experiments).
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