The Transmembrane Domain of Influenza Hemagglutinin Exhibits a Stringent Length Requirement to Support the Hemifusion to Fusion Transition

doi:10.1083/jcb.151.2.425

Published online 16 October 2000. doi:10.1083/jcb.151.2.425

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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

R. Todd Armstrong^a, Anna S. Kushnir^a, and Judith M. White^a

^a Department of Cell Biology, University of Virginia Health System, School of Medicine, Charlottesville, Virginia 22908
Department of Cell Biology, University of Virginia Health System, School of Medicine, P.O. Box 800732, Charlottesville, VA 22908-0732.(804) 982-3912(804) 924-2593

Glycosylphosphatidylinositol-anchored influenza hemagglutinin(GPI-HA) mediates hemifusion, whereas chimeras with foreigntransmembrane (TM) domains mediate full fusion. A possible explanationfor these observations is that the TM domain must be a criticallength in order for HA to promote full fusion. To test thishypothesis, we analyzed biochemical properties and fusion phenotypesof HA with alterations in its 27–amino acid TM domain.Our mutants included sequential 2–amino acid ( ${Delta}$ 2– ${Delta}$ 14) and an 11–amino acid deletion from the COOH-terminalend, deletions of 6 or 8 amino acids from the NH₂-terminal andmiddle regions, and a deletion of 12 amino acids from the NH₂-terminalend of the TM domain. We also made several point mutations inthe TM domain. All of the mutants except ${Delta}$ 14 were expressed atthe cell surface and displayed biochemical properties virtuallyidentical to wild-type HA. All the mutants that were expressedat the cell surface promoted full fusion, with the notable exceptionof deletions of >10 amino acids. A mutant in which 11 aminoacids 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 acidsis needed to efficiently promote full fusion. Addition of eitherthe hydrophilic HA cytoplasmic tail sequence or a single arginineto ${Delta}$ 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 thebilayer to promote full fusion.

Key Words: hemagglutinin • hemifusion • transmembrane domain • glycosylphosphatidylinositol anchor • SNARE proteins

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–sensitivefactor attachment protein receptor; STI, soybean trypsin inhibitor;TM, transmembrane; VSV G, vesicular stomatitis virus envelopeglycoprotein; WT, wild-type.

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