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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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Quantitation of lipid mixing and content mixing. CV-1 cells expressing WT or mutant HAs were prepared for fusion as indicated in Materials and Methods, except that fusion was triggered at pH 5.0 for 2 min at 37°C and reneutralized. The amount of cDNA used per transfection is indicated. Lipid and content mixing events were averaged from 4–12 random fields (mean ± SEM). Percent lipid dye transfer (hatched bars) was determined by dividing the number of cells receiving lipid dye by the number of cells with bound RBCs in each field. Percent content dye transfer (black bars) was determined by dividing the number of cells receiving content dye by the number of cells with bound RBCs in each field. Relative surface expression of HA in fluorescence units (FU), was obtained by FACS® analyses, and is presented as the mean fluorescence intensity per cell normalized to that of 3.5 μg WT HA cDNA.
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Figure 4: Quantitation of lipid mixing and content mixing. CV-1 cells expressing WT or mutant HAs were prepared for fusion as indicated in Materials and Methods, except that fusion was triggered at pH 5.0 for 2 min at 37°C and reneutralized. The amount of cDNA used per transfection is indicated. Lipid and content mixing events were averaged from 4–12 random fields (mean ± SEM). Percent lipid dye transfer (hatched bars) was determined by dividing the number of cells receiving lipid dye by the number of cells with bound RBCs in each field. Percent content dye transfer (black bars) was determined by dividing the number of cells receiving content dye by the number of cells with bound RBCs in each field. Relative surface expression of HA in fluorescence units (FU), was obtained by FACS® analyses, and is presented as the mean fluorescence intensity per cell normalized to that of 3.5 μg WT HA cDNA.

Mentions: We evaluated the fusion activity of Δ2, Δ4, Δ6, Δ8, Δ10, and Δ12 HA using a dye transfer assay. RBCs colabeled with a lipid dye (R18) and a soluble content dye (CF) were bound to HA-expressing cells, and fusion was induced as described in Materials and Methods. After 5 min at 37°C and pH 5, the cells were returned to neutral pH medium and examined with a fluorescence microscope. As seen in Fig. 4, both dyes transferred efficiently to cells expressing Δ2, Δ4, Δ6, Δ8, and Δ10 HA. A different phenotype was seen for cells expressing Δ12 HA: whereas we observed efficient lipid dye transfer (97%), content dye transfer was severely restricted (<10 vs. 97% for WT HA). As expected, we did not observe transfer of R18 or CF by WT or mutant HA-expressing cells at neutral pH or at low pH if the cells had not been pretreated with trypsin to process HA0 (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)

Quantitation of lipid mixing and content mixing. CV-1 cells expressing WT or mutant HAs were prepared for fusion as indicated in Materials and Methods, except that fusion was triggered at pH 5.0 for 2 min at 37°C and reneutralized. The amount of cDNA used per transfection is indicated. Lipid and content mixing events were averaged from 4–12 random fields (mean ± SEM). Percent lipid dye transfer (hatched bars) was determined by dividing the number of cells receiving lipid dye by the number of cells with bound RBCs in each field. Percent content dye transfer (black bars) was determined by dividing the number of cells receiving content dye by the number of cells with bound RBCs in each field. Relative surface expression of HA in fluorescence units (FU), was obtained by FACS® analyses, and is presented as the mean fluorescence intensity per cell normalized to that of 3.5 μg WT HA cDNA.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192652&req=5

Figure 4: Quantitation of lipid mixing and content mixing. CV-1 cells expressing WT or mutant HAs were prepared for fusion as indicated in Materials and Methods, except that fusion was triggered at pH 5.0 for 2 min at 37°C and reneutralized. The amount of cDNA used per transfection is indicated. Lipid and content mixing events were averaged from 4–12 random fields (mean ± SEM). Percent lipid dye transfer (hatched bars) was determined by dividing the number of cells receiving lipid dye by the number of cells with bound RBCs in each field. Percent content dye transfer (black bars) was determined by dividing the number of cells receiving content dye by the number of cells with bound RBCs in each field. Relative surface expression of HA in fluorescence units (FU), was obtained by FACS® analyses, and is presented as the mean fluorescence intensity per cell normalized to that of 3.5 μg WT HA cDNA.
Mentions: We evaluated the fusion activity of Δ2, Δ4, Δ6, Δ8, Δ10, and Δ12 HA using a dye transfer assay. RBCs colabeled with a lipid dye (R18) and a soluble content dye (CF) were bound to HA-expressing cells, and fusion was induced as described in Materials and Methods. After 5 min at 37°C and pH 5, the cells were returned to neutral pH medium and examined with a fluorescence microscope. As seen in Fig. 4, both dyes transferred efficiently to cells expressing Δ2, Δ4, Δ6, Δ8, and Δ10 HA. A different phenotype was seen for cells expressing Δ12 HA: whereas we observed efficient lipid dye transfer (97%), content dye transfer was severely restricted (<10 vs. 97% for WT HA). As expected, we did not observe transfer of R18 or CF by WT or mutant HA-expressing cells at neutral pH or at low pH if the cells had not been pretreated with trypsin to process HA0 (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