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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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Biochemical analyses. (A) Sucrose gradient analysis. Transfected CV-1 cells were trypsin treated, lysed, run on 3–30% continuous sucrose gradients, and fractionated as described in Materials and Methods. The samples were precipitated with Con A–agarose, resolved by 10% SDS-PAGE, and analyzed for HA protein by Western blotting. Processed forms of Δ2–12 HA migrate to a similar position on sucrose gradients as the WT HA trimer (black arrowhead). (B) Conformational change assay. Transfected CV-1 cells were metabolically labeled, trypsin treated, incubated at indicated pH values for 10 min at 37°C, reneutralized, and lysed. Cell lysates were then immunoprecipitated with the C-HA1 antibody, which recognizes only the low pH conformation of HA, resolved by 10% SDS-PAGE, and quantitated by PhosphorImager® analysis. The amount of HA precipitated when the total HA precipitated at pH 5 is considered as 100%. Δ2–12 HA change conformation with a pH dependence similar to WT HA. The results presented for WT HA and Δ2–Δ10 are from a typical experiment. The values given for Δ12 HA are the average from three independent experiments.
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Figure 3: Biochemical analyses. (A) Sucrose gradient analysis. Transfected CV-1 cells were trypsin treated, lysed, run on 3–30% continuous sucrose gradients, and fractionated as described in Materials and Methods. The samples were precipitated with Con A–agarose, resolved by 10% SDS-PAGE, and analyzed for HA protein by Western blotting. Processed forms of Δ2–12 HA migrate to a similar position on sucrose gradients as the WT HA trimer (black arrowhead). (B) Conformational change assay. Transfected CV-1 cells were metabolically labeled, trypsin treated, incubated at indicated pH values for 10 min at 37°C, reneutralized, and lysed. Cell lysates were then immunoprecipitated with the C-HA1 antibody, which recognizes only the low pH conformation of HA, resolved by 10% SDS-PAGE, and quantitated by PhosphorImager® analysis. The amount of HA precipitated when the total HA precipitated at pH 5 is considered as 100%. Δ2–12 HA change conformation with a pH dependence similar to WT HA. The results presented for WT HA and Δ2–Δ10 are from a typical experiment. The values given for Δ12 HA are the average from three independent experiments.

Mentions: We next asked if the mutant HAs form trimers. Processed forms of Δ2–Δ12 HA migrated to a similar position on sucrose gradients as WT HA (Fig. 3 A, arrows). The higher molecular weight band seen in some of the gradients corresponds to intracellular HA0.


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)

Biochemical analyses. (A) Sucrose gradient analysis. Transfected CV-1 cells were trypsin treated, lysed, run on 3–30% continuous sucrose gradients, and fractionated as described in Materials and Methods. The samples were precipitated with Con A–agarose, resolved by 10% SDS-PAGE, and analyzed for HA protein by Western blotting. Processed forms of Δ2–12 HA migrate to a similar position on sucrose gradients as the WT HA trimer (black arrowhead). (B) Conformational change assay. Transfected CV-1 cells were metabolically labeled, trypsin treated, incubated at indicated pH values for 10 min at 37°C, reneutralized, and lysed. Cell lysates were then immunoprecipitated with the C-HA1 antibody, which recognizes only the low pH conformation of HA, resolved by 10% SDS-PAGE, and quantitated by PhosphorImager® analysis. The amount of HA precipitated when the total HA precipitated at pH 5 is considered as 100%. Δ2–12 HA change conformation with a pH dependence similar to WT HA. The results presented for WT HA and Δ2–Δ10 are from a typical experiment. The values given for Δ12 HA are the average from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: Biochemical analyses. (A) Sucrose gradient analysis. Transfected CV-1 cells were trypsin treated, lysed, run on 3–30% continuous sucrose gradients, and fractionated as described in Materials and Methods. The samples were precipitated with Con A–agarose, resolved by 10% SDS-PAGE, and analyzed for HA protein by Western blotting. Processed forms of Δ2–12 HA migrate to a similar position on sucrose gradients as the WT HA trimer (black arrowhead). (B) Conformational change assay. Transfected CV-1 cells were metabolically labeled, trypsin treated, incubated at indicated pH values for 10 min at 37°C, reneutralized, and lysed. Cell lysates were then immunoprecipitated with the C-HA1 antibody, which recognizes only the low pH conformation of HA, resolved by 10% SDS-PAGE, and quantitated by PhosphorImager® analysis. The amount of HA precipitated when the total HA precipitated at pH 5 is considered as 100%. Δ2–12 HA change conformation with a pH dependence similar to WT HA. The results presented for WT HA and Δ2–Δ10 are from a typical experiment. The values given for Δ12 HA are the average from three independent experiments.
Mentions: We next asked if the mutant HAs form trimers. Processed forms of Δ2–Δ12 HA migrated to a similar position on sucrose gradients as WT HA (Fig. 3 A, arrows). The higher molecular weight band seen in some of the gradients corresponds to intracellular HA0.

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