INFLUENZA-VIRUS HEMAGGLUTININ-INDUCED CELL-PLANAR BILAYER FUSION - QUANTITATIVE DISSECTION OF FUSION PORE KINETICS INTO STAGES

Cells expressing the influenza virus hemagglutinin (HA) fuse to planar bilayer membranes under acidic conditions. After an electrically quie scent prefusion stage (Q), a fusion pore forms that enlarges in three subsequent stages. A repetitively flickering pore stage (R) develops i nto a securely open stage (S) that exhibits conductances ranging from a few to tens of nS. The pore then expands to a terminal stage (T) wit h a large conductance on the order of one microSiemens. We have studie d how virus strain, HA receptors in the target bilayer membrane, and c ytoskeleton affect the time a fusion pore remains in each stage. These intervals are referred to as waiting times. In the quiescent stage, w aiting times were very sensitive to the virus strain and presence of g angliosides (HA receptors) in the bilayer. When bilayers contained gan gliosides, the waiting times in the Q stage for cells infected with th e PR/8/34 strain of virus were exponentially distributed, whereas wait ing times for cells infected with the Japan/305/57 strain were not so distributed. Without gangliosides, the waiting time distribution for P R/8/34 infected cells was complex. The waiting times in the R and S st ages of pore growth were exponentially distributed under all tested co nditions. Within the R stage, we analyzed the kinetics of the flickeri ng pore by fitting the open and closed time distributions with a sum o f two exponentials. Neither the open and closed time distributions nor the flickering pore conductance distributions were appreciably affect ed by virus strain or gangliosides. Colchicine and cytochalasin B incr eased the flicker rates, without affecting the waiting time in the R s tage. We conclude that variations in amino acid sequences of the HAs a nd the presence of gangliosides as receptors within the target membran e critically affect the kinetics of fusion pore formation, but do not affect subsequent stages.