The transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition

Glycosylphosphatidylinositol-anchored influenza hemagglutinin (GPI-I-IA) me diates 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 IIA to promote lull f usion. To test this hypothesis, we analyzed biochemical properties acid fus ion phenotypes of HA with alterations in its 27-amino acid TM domain. Our m utants included sequential 2-amino acid (Delta2-Delta 14) and an Il-amino a cid deletion from the COOH-terminal end, deletions of 6 or 8 amino acids fr om the NH2-terminal and middle regions, and a deletion of 12 amino acids fr om the NH2-terminal end of the TM domain. We also made several point mutati ons in the TM domain. All of the mutants except Delta 14 were expressed at the cell surface and displayed biochemical properties virtually identical t o wild-type IIA. All the mutants that were expressed at the cell surface pr omoted full fusion, with the notable exception of deletions of >10 amino ac ids. 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 hydrop hilic HA cytoplasmic tail sequence or a single arginine to 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 w hich the TM domain must span the bilayer to promote full fusion.