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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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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).
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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).

Mentions: LPC inhibits fusion in a wide variety of systems by preventing the merger of contacting lipid monolayer leaflets of membranes (i.e., hemifusion). It is thought to do so because of its positive spontaneous curvature (Chernomordik et al. 1995). Whether or not hemifusion does indeed occur before fusion, in general LPC arrests fusion at the point of membrane merger (Chernomordik et al. 1998) as a lipid constituent of membranes and not by binding to proteins. Stearoyl-LPC (LPC) is particularly useful because it is sufficiently hydrophobic to remain in membranes after any LPC still in solution is removed, or, if necessary, it can be removed from the membranes with a BSA wash (Chernomordik et al. 1997). We added LPC after TAS was established and removed the unbound fraction, using cells labeled with only aqueous dyes so that lipid composition would not be affected by incorporation of lipid dyes. Temperature was raised to 37°C for 15 min, by which time the majority of cell pairs would have fused if LPC had not been added (Fig. 3 A). We then lowered temperature to 23°C, and found that pore formation was meager (Fig. 5 A, second bar). We refer to the stage reached after lowering temperature back to 23°C as a lipid-arrested stage (LAS) of fusion (Fig. 5 A, inset). Fusion still did not occur when the membrane-bound LPC was removed at the point of LAS (data not shown). But fusion occurred efficiently when temperature was again brought to 37°C (Fig. 5 A, third bar); the extent was comparable to the control (first bar) in which LPC had not been added but cells were washed in the same way as if it had. This high level of fusion demonstrates that the action of LPC was reversible and that gp41 was not inactivated when LAS was created. Significantly, blocking fusion (probably at the point of merger of outer leaflets) by the incorporation of LPC into the membrane prevented gp41 from folding into the six-helix bundle: adding T20 (Fig. 5 A, fourth bar) or T21 (fifth bar) after removing the LPC prevented fusion when temperature was raised to 37°C. Mechanistically, the presence of membrane-bound LPC prevented gp41 from transiting into its final and stable six-helix bundle. Thus, we showed that in order for the bundle to form, membrane merger must take place. Therefore, the bundle itself cannot be the cause of membrane merger.


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)

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).
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Related In: Results  -  Collection

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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).
Mentions: LPC inhibits fusion in a wide variety of systems by preventing the merger of contacting lipid monolayer leaflets of membranes (i.e., hemifusion). It is thought to do so because of its positive spontaneous curvature (Chernomordik et al. 1995). Whether or not hemifusion does indeed occur before fusion, in general LPC arrests fusion at the point of membrane merger (Chernomordik et al. 1998) as a lipid constituent of membranes and not by binding to proteins. Stearoyl-LPC (LPC) is particularly useful because it is sufficiently hydrophobic to remain in membranes after any LPC still in solution is removed, or, if necessary, it can be removed from the membranes with a BSA wash (Chernomordik et al. 1997). We added LPC after TAS was established and removed the unbound fraction, using cells labeled with only aqueous dyes so that lipid composition would not be affected by incorporation of lipid dyes. Temperature was raised to 37°C for 15 min, by which time the majority of cell pairs would have fused if LPC had not been added (Fig. 3 A). We then lowered temperature to 23°C, and found that pore formation was meager (Fig. 5 A, second bar). We refer to the stage reached after lowering temperature back to 23°C as a lipid-arrested stage (LAS) of fusion (Fig. 5 A, inset). Fusion still did not occur when the membrane-bound LPC was removed at the point of LAS (data not shown). But fusion occurred efficiently when temperature was again brought to 37°C (Fig. 5 A, third bar); the extent was comparable to the control (first bar) in which LPC had not been added but cells were washed in the same way as if it had. This high level of fusion demonstrates that the action of LPC was reversible and that gp41 was not inactivated when LAS was created. Significantly, blocking fusion (probably at the point of merger of outer leaflets) by the incorporation of LPC into the membrane prevented gp41 from folding into the six-helix bundle: adding T20 (Fig. 5 A, fourth bar) or T21 (fifth bar) after removing the LPC prevented fusion when temperature was raised to 37°C. Mechanistically, the presence of membrane-bound LPC prevented gp41 from transiting into its final and stable six-helix bundle. Thus, we showed that in order for the bundle to form, membrane merger must take place. Therefore, the bundle itself cannot be the cause of membrane merger.

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