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

Melikyan GB, Markosyan RM, Hemmati H, Delmedico MK, Lambert DM, Cohen FS - J. Cell Biol. (2000)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Physiology, Rush Medical College, Chicago, Illinois 60612, USA.

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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(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.
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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.

Mentions: To establish a reference to which TAS could be compared, cells were maintained at 37°C and at a set time were placed on ice to stop further fusion activity. (In control experiments, we verified that this method did arrest further fusion: dye did not spread between additional cells for 1 h after lowering the temperature to 4°C; not shown.) After fusion was stopped, the kinetics of fusion was obtained by microscopically counting the percentage of cell pairs that had exchanged fluorescent dye at a given time (Fig. 3 A, open squares). Fusion was slow: a significant lag time, ∼15–20 min (in agreement with previous studies; Frey et al. 1995; Weiss et al. 1996), preceded the onset of dye spread, and it took ∼2 h to reach its maximum extent.


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

Melikyan GB, Markosyan RM, Hemmati H, Delmedico MK, Lambert DM, Cohen FS - J. Cell Biol. (2000)

(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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192659&req=5

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.
Mentions: To establish a reference to which TAS could be compared, cells were maintained at 37°C and at a set time were placed on ice to stop further fusion activity. (In control experiments, we verified that this method did arrest further fusion: dye did not spread between additional cells for 1 h after lowering the temperature to 4°C; not shown.) After fusion was stopped, the kinetics of fusion was obtained by microscopically counting the percentage of cell pairs that had exchanged fluorescent dye at a given time (Fig. 3 A, open squares). Fusion was slow: a significant lag time, ∼15–20 min (in agreement with previous studies; Frey et al. 1995; Weiss et al. 1996), preceded the onset of dye spread, and it took ∼2 h to reach its maximum extent.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Physiology, Rush Medical College, Chicago, Illinois 60612, USA.

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.

Show MeSH
Related in: MedlinePlus