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© The Rockefeller University Press,
0021-9525/2000//425 $5.00
The Journal of Cell Biology, Volume 151, Number 2,
, 2000 425-438
Original Article |
The Transmembrane Domain of Influenza Hemagglutinin Exhibits a Stringent Length Requirement to Support the Hemifusion to Fusion Transition
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 (
2–14) and an 11–amino acid deletion from the COOH-terminal end, deletions of 6 or 8 amino acids from the NH2-terminal and middle regions, and a deletion of 12 amino acids from the NH2-terminal end of the TM domain. We also made several point mutations in the TM domain. All of the mutants except 14 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 12 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.
Key Words: hemagglutinin • hemifusion • transmembrane domain • glycosylphosphatidylinositol anchor • SNARE proteins
© 2000 The Rockefeller University Press
Abbreviations used in this paper: CF, carboxyfluorescein; CPZ, chlorpromazine; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; MβCD, methyl β-cyclodextrin; RT, room temperature; SCS, supplemented calf serum; SNARE, soluble N-ethylmaleimide–sensitive factor attachment protein receptor; STI, soybean trypsin inhibitor; TM, transmembrane; VSV G, vesicular stomatitis virus envelope glycoprotein; WT, wild-type.
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