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The transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition.

Armstrong RT, Kushnir AS, White JM - J. Cell Biol. (2000)

Bottom Line: We also made several point mutations in the TM domain.All of the mutants except Delta14 were expressed at the cell surface and displayed biochemical properties virtually identical to wild-type HA.Mutants in which 12 amino acids were deleted (from either end) mediated only hemifusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia Health System, School of Medicine, Charlottesville, Virginia 22908, USA.

ABSTRACT
Glycosylphosphatidylinositol-anchored influenza hemagglutinin (GPI-HA) mediates hemifusion, whereas chimeras with foreign transmembrane (TM) domains mediate full fusion. A possible explanation for these observations is that the TM domain must be a critical length in order for HA to promote full fusion. To test this hypothesis, we analyzed biochemical properties and fusion phenotypes of HA with alterations in its 27-amino acid TM domain. Our mutants included sequential 2-amino acid (Delta2-Delta14) and an 11-amino acid deletion from the COOH-terminal end, deletions of 6 or 8 amino acids from the NH(2)-terminal and middle regions, and a deletion of 12 amino acids from the NH(2)-terminal end of the TM domain. We also made several point mutations in the TM domain. All of the mutants except Delta14 were expressed at the cell surface and displayed biochemical properties virtually identical to wild-type HA. All the mutants that were expressed at the cell surface promoted full fusion, with the notable exception of deletions of >10 amino acids. A mutant in which 11 amino acids were deleted was severely impaired in promoting full fusion. Mutants in which 12 amino acids were deleted (from either end) mediated only hemifusion. Hence, a TM domain of 17 amino acids is needed to efficiently promote full fusion. Addition of either the hydrophilic HA cytoplasmic tail sequence or a single arginine to Delta12 HA, the hemifusion mutant that terminates with 15 (hydrophobic) amino acids of the HA TM domain, restored full fusion activity. Our data support a model in which the TM domain must span the bilayer to promote full fusion.

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Effect of CPZ on content mixing. CV-1 cells expressing GPI-HA, NΔ12, and Δ12 HA cDNA were processed for fusion with CF-labeled RBCs in the absence or presence of the indicated amount of CPZ, as described in the legend to Fig. 6. Only 0.5 mM CPZ is able to promote significant content dye transfer (see Fig. 6).
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Figure 7: Effect of CPZ on content mixing. CV-1 cells expressing GPI-HA, NΔ12, and Δ12 HA cDNA were processed for fusion with CF-labeled RBCs in the absence or presence of the indicated amount of CPZ, as described in the legend to Fig. 6. Only 0.5 mM CPZ is able to promote significant content dye transfer (see Fig. 6).

Mentions: Previous work has shown that treatment with 0.1 mM CPZ, a membrane-permeable amphipathic reagent that partitions preferentially into the inner leaflet of the plasma membrane, efficiently induces full fusion in cases of “stunted fusion” caused by performing fusion experiments under suboptimal conditions (Melikyan et al. 1997a). Stunted fusion is operationally defined as lipid mixing without substantial content mixing due to the formation of small or transient fusion pores. It is thought to occur after hemifusion. In contrast, higher concentrations of CPZ (0.4–0.5 mM) are needed to induce GPI-HA to promote content mixing, and the extent of content mixing seen with GPI-HA in the presence of 0.4–0.5 mM CPZ never reaches that seen with WT HA (Melikyan et al. 1997a). Therefore, we assessed the effects of 0.1 and 0.5 mM CPZ on the ability of Δ12 HA, NΔ12 HA, and GPI-HA to promote content transfer. After binding double-labeled RBCs (R18 and CF) to HA-expressing cells, fusion was triggered by lowering the pH to 5.0 for 5 min at 37°C. The medium was reneutralized, and CPZ was added to the cells at neutral pH. After a 1-min incubation at RT, the CPZ solution was replaced with PBS+. The percentage of R18-stained HA-expressing cells that became labeled with CF was then determined. In the absence of CPZ, ∼1, 7, and 3% of cells expressing NΔ12 HA, Δ12 HA, and GPI-HA, respectively, received aqueous dye (Fig. 6). The addition of 0.1 mM CPZ increased content dye transfer to ∼5, 8, and 4%, respectively. Addition of 0.5 mM CPZ induced a greater extent of CF transfer: ∼20, 27, and 22%, respectively. Representative images of aqueous dye transfer before and after the addition of either 0.1 or 0.5 mM CPZ are shown in Fig. 7. Hence, cells expressing NΔ12 HA and Δ12 HA respond similarly to CPZ as do cells expressing GPI-HA in terms of their ability to promote aqueous dye transfer. The presence of R18 in the RBC membrane augments the transfer of aqueous contents to GPI-HA–expressing cells (Markosyan et al. 2000). Therefore, we assessed content dye transfer from RBCs that were not labeled with R18. As expected, content dye transfer under these conditions was less than with double-labeled RBCs (data not shown). Most importantly, Δ12 HA and NΔ12 HA still responded similarly to CPZ as did GPI-HA: a brief treatment with 0.5 but not 0.1 mM CPZ increased content dye transfer (data not shown).


The transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition.

Armstrong RT, Kushnir AS, White JM - J. Cell Biol. (2000)

Effect of CPZ on content mixing. CV-1 cells expressing GPI-HA, NΔ12, and Δ12 HA cDNA were processed for fusion with CF-labeled RBCs in the absence or presence of the indicated amount of CPZ, as described in the legend to Fig. 6. Only 0.5 mM CPZ is able to promote significant content dye transfer (see Fig. 6).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192652&req=5

Figure 7: Effect of CPZ on content mixing. CV-1 cells expressing GPI-HA, NΔ12, and Δ12 HA cDNA were processed for fusion with CF-labeled RBCs in the absence or presence of the indicated amount of CPZ, as described in the legend to Fig. 6. Only 0.5 mM CPZ is able to promote significant content dye transfer (see Fig. 6).
Mentions: Previous work has shown that treatment with 0.1 mM CPZ, a membrane-permeable amphipathic reagent that partitions preferentially into the inner leaflet of the plasma membrane, efficiently induces full fusion in cases of “stunted fusion” caused by performing fusion experiments under suboptimal conditions (Melikyan et al. 1997a). Stunted fusion is operationally defined as lipid mixing without substantial content mixing due to the formation of small or transient fusion pores. It is thought to occur after hemifusion. In contrast, higher concentrations of CPZ (0.4–0.5 mM) are needed to induce GPI-HA to promote content mixing, and the extent of content mixing seen with GPI-HA in the presence of 0.4–0.5 mM CPZ never reaches that seen with WT HA (Melikyan et al. 1997a). Therefore, we assessed the effects of 0.1 and 0.5 mM CPZ on the ability of Δ12 HA, NΔ12 HA, and GPI-HA to promote content transfer. After binding double-labeled RBCs (R18 and CF) to HA-expressing cells, fusion was triggered by lowering the pH to 5.0 for 5 min at 37°C. The medium was reneutralized, and CPZ was added to the cells at neutral pH. After a 1-min incubation at RT, the CPZ solution was replaced with PBS+. The percentage of R18-stained HA-expressing cells that became labeled with CF was then determined. In the absence of CPZ, ∼1, 7, and 3% of cells expressing NΔ12 HA, Δ12 HA, and GPI-HA, respectively, received aqueous dye (Fig. 6). The addition of 0.1 mM CPZ increased content dye transfer to ∼5, 8, and 4%, respectively. Addition of 0.5 mM CPZ induced a greater extent of CF transfer: ∼20, 27, and 22%, respectively. Representative images of aqueous dye transfer before and after the addition of either 0.1 or 0.5 mM CPZ are shown in Fig. 7. Hence, cells expressing NΔ12 HA and Δ12 HA respond similarly to CPZ as do cells expressing GPI-HA in terms of their ability to promote aqueous dye transfer. The presence of R18 in the RBC membrane augments the transfer of aqueous contents to GPI-HA–expressing cells (Markosyan et al. 2000). Therefore, we assessed content dye transfer from RBCs that were not labeled with R18. As expected, content dye transfer under these conditions was less than with double-labeled RBCs (data not shown). Most importantly, Δ12 HA and NΔ12 HA still responded similarly to CPZ as did GPI-HA: a brief treatment with 0.5 but not 0.1 mM CPZ increased content dye transfer (data not shown).

Bottom Line: We also made several point mutations in the TM domain.All of the mutants except Delta14 were expressed at the cell surface and displayed biochemical properties virtually identical to wild-type HA.Mutants in which 12 amino acids were deleted (from either end) mediated only hemifusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia Health System, School of Medicine, Charlottesville, Virginia 22908, USA.

ABSTRACT
Glycosylphosphatidylinositol-anchored influenza hemagglutinin (GPI-HA) mediates hemifusion, whereas chimeras with foreign transmembrane (TM) domains mediate full fusion. A possible explanation for these observations is that the TM domain must be a critical length in order for HA to promote full fusion. To test this hypothesis, we analyzed biochemical properties and fusion phenotypes of HA with alterations in its 27-amino acid TM domain. Our mutants included sequential 2-amino acid (Delta2-Delta14) and an 11-amino acid deletion from the COOH-terminal end, deletions of 6 or 8 amino acids from the NH(2)-terminal and middle regions, and a deletion of 12 amino acids from the NH(2)-terminal end of the TM domain. We also made several point mutations in the TM domain. All of the mutants except Delta14 were expressed at the cell surface and displayed biochemical properties virtually identical to wild-type HA. All the mutants that were expressed at the cell surface promoted full fusion, with the notable exception of deletions of >10 amino acids. A mutant in which 11 amino acids were deleted was severely impaired in promoting full fusion. Mutants in which 12 amino acids were deleted (from either end) mediated only hemifusion. Hence, a TM domain of 17 amino acids is needed to efficiently promote full fusion. Addition of either the hydrophilic HA cytoplasmic tail sequence or a single arginine to Delta12 HA, the hemifusion mutant that terminates with 15 (hydrophobic) amino acids of the HA TM domain, restored full fusion activity. Our data support a model in which the TM domain must span the bilayer to promote full fusion.

Show MeSH
Related in: MedlinePlus