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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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Additional TM domain deletion and point mutants. Line diagram of the HA gene. In detail is the region surrounding the HA TM domain (gray box). Point mutations were made in the context of full-length HA and are indicated in large font. Deletion mutations were made in the context of tail− HA and are indicated as spaces. Δ10, Δ12, and NΔ12 HA are included as a reference.
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Figure 11: Additional TM domain deletion and point mutants. Line diagram of the HA gene. In detail is the region surrounding the HA TM domain (gray box). Point mutations were made in the context of full-length HA and are indicated in large font. Deletion mutations were made in the context of tail− HA and are indicated as spaces. Δ10, Δ12, and NΔ12 HA are included as a reference.

Mentions: To ascertain whether we could detect any specific TM domain sequences needed for HA to promote full fusion, we constructed additional point and deletion mutations within the HA TM domain (Fig. 11). We mutated the tryptophans (to alanines) within the highly conserved WILW sequence at the beginning of the HA TM domain. We mutated a serine at position 194, since this residue is the analogue of a glycine implicated as being important for the fusion activity of Japan HA (Melikyan et al. 1999). We also mutated a glycine at position 204 (singly and in combination with Ser 194), since glycines near the middle of the TM domain have been reported to be important for fusion mediated by the vesicular stomatitis virus envelope glycoprotein (VSV G; Cleverley and Lenard 1998). We also deleted six or eight amino acids at different locations within the TM domain. All mutants were examined for expression at the cell surface, for their ability to be cleaved by trypsin into HA1 and HA2, for RBC binding, and for fusion (both lipid and content mixing). Most of the mutants were also examined for trimer formation and the pH dependence of the conformation change (Table ). As seen in Table , by all of the criteria examined, these additional mutants in the HA TM domain behaved virtually the same as WT HA. Most importantly, all of the 12 additional HA TM domain mutants exhibited efficient lipid and efficient content mixing.


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)

Additional TM domain deletion and point mutants. Line diagram of the HA gene. In detail is the region surrounding the HA TM domain (gray box). Point mutations were made in the context of full-length HA and are indicated in large font. Deletion mutations were made in the context of tail− HA and are indicated as spaces. Δ10, Δ12, and NΔ12 HA are included as a reference.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 11: Additional TM domain deletion and point mutants. Line diagram of the HA gene. In detail is the region surrounding the HA TM domain (gray box). Point mutations were made in the context of full-length HA and are indicated in large font. Deletion mutations were made in the context of tail− HA and are indicated as spaces. Δ10, Δ12, and NΔ12 HA are included as a reference.
Mentions: To ascertain whether we could detect any specific TM domain sequences needed for HA to promote full fusion, we constructed additional point and deletion mutations within the HA TM domain (Fig. 11). We mutated the tryptophans (to alanines) within the highly conserved WILW sequence at the beginning of the HA TM domain. We mutated a serine at position 194, since this residue is the analogue of a glycine implicated as being important for the fusion activity of Japan HA (Melikyan et al. 1999). We also mutated a glycine at position 204 (singly and in combination with Ser 194), since glycines near the middle of the TM domain have been reported to be important for fusion mediated by the vesicular stomatitis virus envelope glycoprotein (VSV G; Cleverley and Lenard 1998). We also deleted six or eight amino acids at different locations within the TM domain. All mutants were examined for expression at the cell surface, for their ability to be cleaved by trypsin into HA1 and HA2, for RBC binding, and for fusion (both lipid and content mixing). Most of the mutants were also examined for trimer formation and the pH dependence of the conformation change (Table ). As seen in Table , by all of the criteria examined, these additional mutants in the HA TM domain behaved virtually the same as WT HA. Most importantly, all of the 12 additional HA TM domain mutants exhibited efficient lipid and efficient content mixing.

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