Limits...
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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Related in: MedlinePlus

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).
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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).

Mentions: The extent of fusion was assessed by visual microscopic examination. As required, the fluorescence images were captured with an intensified (KS1380; Video Scope) CCD video camera (model 72; Dage-MTI, Inc.), were recorded on S-VHS format videotape, and the images were analyzed off-line (Qiao et al. 1999). Images of fluorescent cells were acquired separately for each fluorescent dye, digitized, and pseudocolored according to their emission wavelength. For cells bound to polylysine-coated glass, three images were then superimposed to detect whether fluorescent dyes had redistributed; fused cells had a lighter (pinkish) appearance. Cells partially overlapping in the bound state also present a lighter appearance, but are readily distinguishable from cells that have actually fused. The fraction of effector and target cells in contact that became stained with all 3 dyes (screening >100 cell pairs per well) provided the extent of fusion (Melikyan et al. 2000). For example, ∼50% of the contacting cells have fused in Fig. 2 B. When effector cells were placed upon target cells grown on untreated glass, the two images (one for each aqueous dye) were superimposed. The extent of fusion was quantified as the number of target cells that acquired calcein (from the effector cells) normalized by the total number of target cells in the field of view. When high-time resolution kinetic measurements for the onset of calcein spread between effector and target cells were required, a temperature jump (T-jump) technique was used (see below) to determine the cells that fused within 5 min of raising temperature (∼20% of the total cell pairs).


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 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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192659&req=5

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).
Mentions: The extent of fusion was assessed by visual microscopic examination. As required, the fluorescence images were captured with an intensified (KS1380; Video Scope) CCD video camera (model 72; Dage-MTI, Inc.), were recorded on S-VHS format videotape, and the images were analyzed off-line (Qiao et al. 1999). Images of fluorescent cells were acquired separately for each fluorescent dye, digitized, and pseudocolored according to their emission wavelength. For cells bound to polylysine-coated glass, three images were then superimposed to detect whether fluorescent dyes had redistributed; fused cells had a lighter (pinkish) appearance. Cells partially overlapping in the bound state also present a lighter appearance, but are readily distinguishable from cells that have actually fused. The fraction of effector and target cells in contact that became stained with all 3 dyes (screening >100 cell pairs per well) provided the extent of fusion (Melikyan et al. 2000). For example, ∼50% of the contacting cells have fused in Fig. 2 B. When effector cells were placed upon target cells grown on untreated glass, the two images (one for each aqueous dye) were superimposed. The extent of fusion was quantified as the number of target cells that acquired calcein (from the effector cells) normalized by the total number of target cells in the field of view. When high-time resolution kinetic measurements for the onset of calcein spread between effector and target cells were required, a temperature jump (T-jump) technique was used (see below) to determine the cells that fused within 5 min of raising temperature (∼20% of the total cell pairs).

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