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 rec ombinant Virus was used to express wild-type (WT) HA and HA mutants co ntaining changes in the HA cytoplasmic tail. HA and its mutants were e xpressed in CV-1 cells and the ability of these cells to fuse to eithe r red blood cells (RBCs) or planar bilayer membranes was determined qu antitatively. The percentage oi 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 cystein e residues were replaced to block the addition of palmitate. When RBCs were colabeled with large and small aqueous dyes and fused to CV-1 ce lls expressing WT HA transfer of the large dye was significantly slowe r and extent of transfer was lower;han that of the small dye, indicati ng 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 e ither dye. When CV-I cells expressing WT HA were fused to planar membr anes, small pores tended to open and close repetitively (''flicker'') before a pore would continue to either grow irreversibly to large cond uctances or grow to intermediate sizes and then contract. For HA mutan ts CT-and MAY, flickering was less likely to occur, but these pores di d evolve in a manner identical to WT HA postflicker pores. We conclude that palmitate covalently linked to cysteine residues of the HA cytop lasmic tail is required for pore flickering, but that the tail does no t play an important role in subsequent pore enlargement. (C) 1997 Acad emic Press.