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. 2000 Oct 16;151(2):413-23.
doi: 10.1083/jcb.151.2.413.

Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion

G B Melikyan  1 , R M Markosyan, H Hemmati, M K Delmedico, D M Lambert, F S Cohen
Affiliations

Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion

G B Melikyan et al. J Cell Biol. .

Abstract

Many viral fusion proteins exhibit a six-helix bundle as a core structure. HIV Env-induced fusion was studied to resolve whether membrane merger was due to the transition into the bundle configuration or occurred after bundle formation. Suboptimal temperature was used to arrest fusion at an intermediate stage. When bundle formation was prevented by adding inhibitory peptides at this stage, membranes did not merge upon raising temperature. Inversely, when membrane merger was prevented by incorporating lysophosphatidylcholine (LPC) into cell membranes at the intermediate, the bundle did not form upon optimizing temperature. In the absence of LPC, the six-helix bundle did not form when the temperature of the intermediate was raised for times too short to promote fusion. Kinetic measures showed that after the temperature pulse, cells had not advanced further toward fusion. The latter results indicate that bundle formation is the rate-limiting step between the arrested intermediate and fusion. Electrical measures showed that the HIV Env-induced pore is initially large and grows rapidly. It is proposed that bundle formation and fusion are each contingent on the other and that movement of Env during its transition into the six-helix bundle directly induces the lipid rearrangements of membrane fusion. Because peptide inhibition showed that, at the intermediate stage, the heptad repeats of gp41 have become stably exposed, creation of the intermediate could be of importance in drug and/or vaccine development.

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Figures

Figure 1
Figure 1
Schematic of location of HR1 and HR2 within HIV-1 gp41 and the hairpin bend of a monomer within a six-helix bundle. FP, NH2-terminal fusion peptide; TM, transmembrane domain.
Figure 3
Figure 3
(A) The fraction of cells in contact that fuse as a function of time at 37°C. Cells were either coincubated directly at 37°C (□), or TAS (▴) or 4°-TAS (○) was first established and temperature was then raised to 37°C. Error bars show the standard error of four to eight experiments. (B) The ability of agents to block fusion through binding to gp120, CD4, or CXCR4. In control experiments (first bar), TAS was created and temperature was then brought to 37°C for 45 min. sCD4, a neutralizing antibody against CD4 (Q4120), or a peptide that binds to CXCR4 (T22) were added either at the beginning of coincubating the effector and target cells for 3 h at 23°C (23°C, cross-hatched bars) or after establishing TAS by a 2-h coincubation and then allowing 1 h at 23°C for the agents to bind (black bars). Alternatively, the inhibitory agents were added either before (4°C, cross-hatched bars) or after a 2-h coincubation (establishing 4°-TAS) of cells at 4°C (black bars). The extent of fusion was normalized by the control experiments without inhibitory agents. The concentrations of the agents were: 50 μg/ml of sCD4, 40 μg/ml of Q4120, and 20 nM of T22 peptide.
Figure 2
Figure 2
A three-color fluorescent assay of fusion. The cells were adhered to polylysine-coated glass. Effector cells are green (calcein) and target cells are purple (a mixture of the red DiI and the blue CMAC). Fused cells are pinkish. (A) Fusion did not occur at TAS. (B) After raising the temperature to 37°C for 30 min, fusion occurred and all three dyes redistributed (arrowheads).
Figure 4
Figure 4
Using T20 and T21 to assess formation of six-helix bundles. (A) T20 and T21 inhibit fusion after sCD4 binding in the absence of target cells. Effector TF228 cells were treated with agents as indicated and the agents were removed by washing; the effector cells were then bound to target HelaT4 cells grown on untreated glass, and fusion was measured after raising temperature to 37°C for 2 h. When adding T20 and T21 to effector cells alone, fusion occurred to the same extent as for the control (first bar). Adding sCD4 alone did not affect fusion (sixth bar), but the addition did expose the HRs to T20 (third versus second bar) and T21 (fifth versus fourth bar). The peptides and sCD4 were added simultaneously. (B) T20 and T21 inhibited fusion after creating TAS and the peptides remained bound to gp41 for long times. Effector cells were laid on top of target cells grown on slides and TAS was created. After a mock wash (without adding peptides), raising temperature to 37°C for 30 min led to fusion (first bar). (The extent of fusion was the same when the mock wash was omitted.) Adding T20 or T21 after TAS was created abolished fusion (second and third bars). Fusion was significantly reduced even after washing out unbound T20 or T21 and immediately raising temperature to 37°C (fourth and fifth cross-hatched bars). Raising temperature 1 h after washing led to a greater, but still significantly impeded, extent of fusion (white bars above cross-hatches). In control experiments, T20 or T21 (black bars) were incubated for 1 h at 23°C with target cells grown on slides, the unbound peptides were removed by washing, the effector cells were bound, and temperature was raised to 37°C for 2 h, fusion was not inhibited. The concentrations of agents were: 50 μg/ml of sCD4, 40 nM T20, and 220 nM T21.
Figure 5
Figure 5
The formation of the six-helix bundle requires membrane merger. (A) Adding 285 μM LPC to cells at TAS, followed by removal of the unbound LPC 5 min later, suppressed fusion when temperature was raised to 37°C (LAS, second bar). But removing the membrane-bound LPC allowed fusion to occur at 37°C (third bar). Addition of 40 nM T20 or 440 nM T21 after removing all LPC inhibited fusion when temperature was again increased to 37°C (fourth and fifth bars). Effector cells were bound to target cells that had been grown overnight on untreated glass. (B) Exposure of cells at TAS to 265 μM OA yielded the same extent of fusion (second bar) as the control (first bar). LPC was added to cells at TAS and the unbound fraction was removed (with the cells retaining the membrane-bound LPC); the addition of T20 or T21 (concentrations as in A) followed by removal of the membrane-bound LPC and unbound peptide suppressed fusion (third and fourth bars).
Figure 6
Figure 6
Fusion evoked from TAS by a T-jump to 37°C. (A) Fluorescence images of fusion between calcein-labeled effector cells and unlabeled target cells (the bright field image is superimposed on the first panel). The times after the T-jump are shown. Arrowhead indicates the first target cell that acquired aqueous dye. Effector and target cells were mixed together and plated on polylysine-coated coverslips. (B) The extents of fusion for preconditioned cells in the absence and presence of peptides. TAS was created and temperature was then decreased to 4°C. Preconditioned cells were generated by increasing the temperature of several cells in a microscopic view to 37°C and once one or two cells fused, quickly lowering the temperature to 4°C. T20 (middle bar) or T21 (right bar) were added or not added (left bar, control) and fusion of the remaining preconditioned cells was induced by again raising temperature to 37°C. Inset: kinetics of fusion when temperature was maintained at 37°C was continuously monitored either by the onset of calcein redistribution (○) or by measuring the increments in electrical capacitance of cell membranes due to fusion pore opening (•). Fusion kinetics of preconditioned cells measured by calcein redistribution is shown by ▵. (C) Electrical recording of fusion pores formed by Env. A representative pore is shown in the inset. The average pore conductance was determined from eight individual records (•). The right-hand scale shows the approximate pore diameter. Bars indicate the standard error.
Figure 7
Figure 7
A proposed sequence of events for Env-mediated membrane fusion. The relative positions of HR1 and HR2 in the bound state are not known and only HR1 is depicted. (Neither CD4 nor the coreceptors are shown.) When gp41 is activated, the grooves of the central coiled coil (light gray bars with fusion peptide indicated by arrows) and the COOH-terminal helices (dark gray bars attached to the TM domain and cytoplasmic tail) have become exposed. The gp120 on the left is made transparent, for clarity. When gp41 further reconfigures into a six-helix bundle, the fusion peptide and TM domain (in different membranes) are forced toward each other and induce pore formation (probably by rupturing the hemifusion diaphragm, not shown).
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