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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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Processing of HA truncation mutants. (A) Chymotrypsin (C) or trypsin (T) treated CV-1 cells were biotinylated, lysed, immunoprecipitated with the Site A antibody, resolved by SDS-PAGE, transferred to nitrocellulose, and visualized with streptavidin-HRP. Like WT HA, all mutant HAs exhibit efficient processing. (B) CV-1 cells were metabolically labeled, treated with trypsin, lysed, immunoprecipitated with an HA-specific mAb, treated with 1 U of N-Glycosidase F for 2 h at 37°C, and resolved on 15% SDS-PAGE. The gels were dried and exposed to film. Δ2–12 HA exhibit a mobility shift indicative of a truncated HA2 subunit (HA2*).
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Figure 2: Processing of HA truncation mutants. (A) Chymotrypsin (C) or trypsin (T) treated CV-1 cells were biotinylated, lysed, immunoprecipitated with the Site A antibody, resolved by SDS-PAGE, transferred to nitrocellulose, and visualized with streptavidin-HRP. Like WT HA, all mutant HAs exhibit efficient processing. (B) CV-1 cells were metabolically labeled, treated with trypsin, lysed, immunoprecipitated with an HA-specific mAb, treated with 1 U of N-Glycosidase F for 2 h at 37°C, and resolved on 15% SDS-PAGE. The gels were dried and exposed to film. Δ2–12 HA exhibit a mobility shift indicative of a truncated HA2 subunit (HA2*).

Mentions: We initially made a set of mutants in which we sequentially deleted 2, 4, 6, 8, 10, 12, and 14 amino acids from the COOH-terminal end of the HA TM domain in the context of a Tail− HA construct (Fig. 1). We first asked whether these mutant HAs could be expressed at the cell surface in a fusion-permissive form (i.e., cleaved from HA0 to HA1-S-S-HA2). We also examined them for a shift in the migration of their HA2 subunits. With the exception of Δ14 (data not shown), all of the mutants were expressed at the cell surface as HA0 and were efficiently cleaved to HA1 and HA2 by the addition of trypsin (Fig. 2 A). Δ2–Δ8 HA exhibited the expected shift in the mobility of the HA2 subunits (Fig. 2 B, left). Δ10 and Δ12 HA exhibited an increased mobility compared with WT HA (and Tail− HA; data not shown), but these mutants migrated more slowly than Δ8 HA (Fig. 2 B, right). The fact that Δ12 was, but that Δ14 was not, expressed at the cell surface is consistent with previous observations that a mutant HA with a 13–amino acid truncation of the TM domain was not transported beyond the cis-Golgi compartment (Doyle et al. 1986).


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)

Processing of HA truncation mutants. (A) Chymotrypsin (C) or trypsin (T) treated CV-1 cells were biotinylated, lysed, immunoprecipitated with the Site A antibody, resolved by SDS-PAGE, transferred to nitrocellulose, and visualized with streptavidin-HRP. Like WT HA, all mutant HAs exhibit efficient processing. (B) CV-1 cells were metabolically labeled, treated with trypsin, lysed, immunoprecipitated with an HA-specific mAb, treated with 1 U of N-Glycosidase F for 2 h at 37°C, and resolved on 15% SDS-PAGE. The gels were dried and exposed to film. Δ2–12 HA exhibit a mobility shift indicative of a truncated HA2 subunit (HA2*).
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Related In: Results  -  Collection

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

Figure 2: Processing of HA truncation mutants. (A) Chymotrypsin (C) or trypsin (T) treated CV-1 cells were biotinylated, lysed, immunoprecipitated with the Site A antibody, resolved by SDS-PAGE, transferred to nitrocellulose, and visualized with streptavidin-HRP. Like WT HA, all mutant HAs exhibit efficient processing. (B) CV-1 cells were metabolically labeled, treated with trypsin, lysed, immunoprecipitated with an HA-specific mAb, treated with 1 U of N-Glycosidase F for 2 h at 37°C, and resolved on 15% SDS-PAGE. The gels were dried and exposed to film. Δ2–12 HA exhibit a mobility shift indicative of a truncated HA2 subunit (HA2*).
Mentions: We initially made a set of mutants in which we sequentially deleted 2, 4, 6, 8, 10, 12, and 14 amino acids from the COOH-terminal end of the HA TM domain in the context of a Tail− HA construct (Fig. 1). We first asked whether these mutant HAs could be expressed at the cell surface in a fusion-permissive form (i.e., cleaved from HA0 to HA1-S-S-HA2). We also examined them for a shift in the migration of their HA2 subunits. With the exception of Δ14 (data not shown), all of the mutants were expressed at the cell surface as HA0 and were efficiently cleaved to HA1 and HA2 by the addition of trypsin (Fig. 2 A). Δ2–Δ8 HA exhibited the expected shift in the mobility of the HA2 subunits (Fig. 2 B, left). Δ10 and Δ12 HA exhibited an increased mobility compared with WT HA (and Tail− HA; data not shown), but these mutants migrated more slowly than Δ8 HA (Fig. 2 B, right). The fact that Δ12 was, but that Δ14 was not, expressed at the cell surface is consistent with previous observations that a mutant HA with a 13–amino acid truncation of the TM domain was not transported beyond the cis-Golgi compartment (Doyle et al. 1986).

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