The role of the cytoplasmic tail region of influenza virus hemagglutinin in formation and growth of fusion pores

The effect of the cytoplasmic tail of influenza hemagglutinin (HA) (H3 subtype) on fusion kinetics and pore growth was examined. An SV40 recombinant virus was used to express wild-type (WT) HA and HA mutants containing changes in the HA cytoplasmic tail HA and its mutants were expressed in CV-1 cells and the ability of these cells to fuse to either red blood cells (RBCs) or planar bilayer membranes was determined quantitatively. The percentage of cells expressing HA and the levels of expression were the same for WT HA or HA lacking its cytoplasmic tail (CT^- ), and for a mutant. MAY, in which the three HA C-terminal cysteine residues were replaced to block the addition of palmitate. When RBCs were colabeled with large and small aqueous dyes and fused to CV-1 cells expressing WT HA, transfer of the large dye was significantly slower and extent of transfer was lower than that of the small dye, indicating that pores did not expand quickly to large diameters. An absence of the HA cytoplasmic tail did not alter the time course of spread for either dye. When CV-1 cells expressing WT HA were fused to planar membranes, small pores tended to open and close repetitively ('flicker') before a pore would continue to either grow irreversibly to large conductances or grow to intermediate sizes and then contract. For HA mutants CT^- and MAY, flickering was less likely to occur, but these pores did evolve in a manner identical to WT HA postflicker pores. We conclude that palmitate covalently linked to cysteine residues of the HA cytoplasmic tail is required for pore flickering, but that the tail does not play an important role in subsequent pore enlargement.