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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 carbonate extraction and cholesterol depletion on HA TM mutants. (A) Carbonate extraction. Microsomal membranes were prepared, adjusted to pH 11.0, and HA in the supernatant and pellet fractions was prepared, immunoprecipitated with an anti-HA mAb, and detected as described in Materials and Methods. Processed HA (WT and mutant) was only found in the pellet fraction, indicating that it is not extracted by high pH. (B) Triton X-100 insolubility. CV-1 cells expressing WT or mutant HAs were incubated in the absence (−) or the presence (+) of the cholesterol-depleting reagent MβCD (20 mM) for 30 min at 37°C. HA was prepared and divided into insoluble and soluble fractions and detected as described in Materials and Methods. The percentage of HA found in the insoluble fraction in the absence or presence of MβCD was determined. Cholesterol depletion by treatment with MβCD increases the Triton X-100 solubility of WT HA and GPI-HA, but not of Δ12 HA (n = 4). (C) Effect of cholesterol depletion on fusion. CV-1 cells expressing WT or mutant HAs were prepared as described in the legend to Fig. 4, depleted of cholesterol as described in B, bound to labeled RBCs, and triggered for fusion. Cholesterol depletion by treatment with MβCD does not affect the fusion phenotype of WT HA, GPI-HA, Δ12 HA, or NΔ12 HA (see Fig. 5).
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Figure 8: Effect of carbonate extraction and cholesterol depletion on HA TM mutants. (A) Carbonate extraction. Microsomal membranes were prepared, adjusted to pH 11.0, and HA in the supernatant and pellet fractions was prepared, immunoprecipitated with an anti-HA mAb, and detected as described in Materials and Methods. Processed HA (WT and mutant) was only found in the pellet fraction, indicating that it is not extracted by high pH. (B) Triton X-100 insolubility. CV-1 cells expressing WT or mutant HAs were incubated in the absence (−) or the presence (+) of the cholesterol-depleting reagent MβCD (20 mM) for 30 min at 37°C. HA was prepared and divided into insoluble and soluble fractions and detected as described in Materials and Methods. The percentage of HA found in the insoluble fraction in the absence or presence of MβCD was determined. Cholesterol depletion by treatment with MβCD increases the Triton X-100 solubility of WT HA and GPI-HA, but not of Δ12 HA (n = 4). (C) Effect of cholesterol depletion on fusion. CV-1 cells expressing WT or mutant HAs were prepared as described in the legend to Fig. 4, depleted of cholesterol as described in B, bound to labeled RBCs, and triggered for fusion. Cholesterol depletion by treatment with MβCD does not affect the fusion phenotype of WT HA, GPI-HA, Δ12 HA, or NΔ12 HA (see Fig. 5).

Mentions: Given the striking phenotype of HA lacking 12 amino acids in the TM domain (lipid, but not content, mixing), we explored how Δ12 HA is anchored in the membrane. Like WT HA, Δ12 HA (as well as GPI-HA) was resistant to carbonate extraction (Fig. 8 A). Given that some GPI-anchored proteins associate with cholesterol and sphingomyelin-rich detergent-insoluble membrane fractions (DIGs; Simons and Ikonen 1997; Friedrichson and Kurzchalia 1998; Varma and Mayor 1998), we examined the solubility of Δ12 HA and NΔ12 HA in Triton X-100 at 4°C before and after treating cells with methyl β-cyclodextrin to remove cholesterol (Scheiffele et al. 1997). Proteins that associate with lipid raft microdomains are often relatively insoluble in 1% Triton X-100 in the cold (Simons and Ikonen 1997). Cholesterol depletion can increase the solubility of these proteins in Triton X-100, presumably by disrupting the raft microdomains (Scheiffele et al. 1997). Both Δ12 HA and NΔ12 HA (data not shown) were readily solubilized by Triton X-100 at 4°C, suggesting that they do not associate with DIGs. In contrast, both WT HA and GPI-HA were partially insoluble in Triton X-100 at 4°C, and depletion of cholesterol appeared to increase their solubility (Fig. 8 B).


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 carbonate extraction and cholesterol depletion on HA TM mutants. (A) Carbonate extraction. Microsomal membranes were prepared, adjusted to pH 11.0, and HA in the supernatant and pellet fractions was prepared, immunoprecipitated with an anti-HA mAb, and detected as described in Materials and Methods. Processed HA (WT and mutant) was only found in the pellet fraction, indicating that it is not extracted by high pH. (B) Triton X-100 insolubility. CV-1 cells expressing WT or mutant HAs were incubated in the absence (−) or the presence (+) of the cholesterol-depleting reagent MβCD (20 mM) for 30 min at 37°C. HA was prepared and divided into insoluble and soluble fractions and detected as described in Materials and Methods. The percentage of HA found in the insoluble fraction in the absence or presence of MβCD was determined. Cholesterol depletion by treatment with MβCD increases the Triton X-100 solubility of WT HA and GPI-HA, but not of Δ12 HA (n = 4). (C) Effect of cholesterol depletion on fusion. CV-1 cells expressing WT or mutant HAs were prepared as described in the legend to Fig. 4, depleted of cholesterol as described in B, bound to labeled RBCs, and triggered for fusion. Cholesterol depletion by treatment with MβCD does not affect the fusion phenotype of WT HA, GPI-HA, Δ12 HA, or NΔ12 HA (see Fig. 5).
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Figure 8: Effect of carbonate extraction and cholesterol depletion on HA TM mutants. (A) Carbonate extraction. Microsomal membranes were prepared, adjusted to pH 11.0, and HA in the supernatant and pellet fractions was prepared, immunoprecipitated with an anti-HA mAb, and detected as described in Materials and Methods. Processed HA (WT and mutant) was only found in the pellet fraction, indicating that it is not extracted by high pH. (B) Triton X-100 insolubility. CV-1 cells expressing WT or mutant HAs were incubated in the absence (−) or the presence (+) of the cholesterol-depleting reagent MβCD (20 mM) for 30 min at 37°C. HA was prepared and divided into insoluble and soluble fractions and detected as described in Materials and Methods. The percentage of HA found in the insoluble fraction in the absence or presence of MβCD was determined. Cholesterol depletion by treatment with MβCD increases the Triton X-100 solubility of WT HA and GPI-HA, but not of Δ12 HA (n = 4). (C) Effect of cholesterol depletion on fusion. CV-1 cells expressing WT or mutant HAs were prepared as described in the legend to Fig. 4, depleted of cholesterol as described in B, bound to labeled RBCs, and triggered for fusion. Cholesterol depletion by treatment with MβCD does not affect the fusion phenotype of WT HA, GPI-HA, Δ12 HA, or NΔ12 HA (see Fig. 5).
Mentions: Given the striking phenotype of HA lacking 12 amino acids in the TM domain (lipid, but not content, mixing), we explored how Δ12 HA is anchored in the membrane. Like WT HA, Δ12 HA (as well as GPI-HA) was resistant to carbonate extraction (Fig. 8 A). Given that some GPI-anchored proteins associate with cholesterol and sphingomyelin-rich detergent-insoluble membrane fractions (DIGs; Simons and Ikonen 1997; Friedrichson and Kurzchalia 1998; Varma and Mayor 1998), we examined the solubility of Δ12 HA and NΔ12 HA in Triton X-100 at 4°C before and after treating cells with methyl β-cyclodextrin to remove cholesterol (Scheiffele et al. 1997). Proteins that associate with lipid raft microdomains are often relatively insoluble in 1% Triton X-100 in the cold (Simons and Ikonen 1997). Cholesterol depletion can increase the solubility of these proteins in Triton X-100, presumably by disrupting the raft microdomains (Scheiffele et al. 1997). Both Δ12 HA and NΔ12 HA (data not shown) were readily solubilized by Triton X-100 at 4°C, suggesting that they do not associate with DIGs. In contrast, both WT HA and GPI-HA were partially insoluble in Triton X-100 at 4°C, and depletion of cholesterol appeared to increase their solubility (Fig. 8 B).

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