PROBE TRANSFER WITH AND WITHOUT MEMBRANE-FUSION IN A FLUORESCENCE FUSION ASSAY

An analysis of the R-18 fusion assay was made during the fusion of the Sendai virus with erythrocyte ghosts. The increase in R-18 fluorescen ce, reflecting the interaction process, was evaluated in terms of the different processes that in principle may contribute to this increase, that is, monomeric probe transfer, hemifusion, and complete fusion. T o this end, the kinetics of the R-18-labeled lipid mixing were compare d to those obtained with an assay in which the fusion-monitoring probe , eosin-maleimide, was attached to the viral surface proteins. The exp eriments relied on the use of native and fusion-inactive viruses and s tudies involving viral and target membranes that were modified by the incorporation of the lysophospholipid. The total dequenching signal de tected in the R-18 assay consists of components from probe transferred without fusion and from fusion itself. At 37 degrees C, the initial r ate of dequenching (within two minutes) was predominately from the pro be diluted by fusion with little contribution from transfer. The deque nching signal due to the probe transfer without fusion occurred at tem peratures as low as 10 degrees C and increased linearly with time. Com plete fusion started at about 20-25 degrees C and increased sharply at 30 degrees C. The extent of hemifusion was deduced from the total R-1 8 dequenching data and those of the eosin-maleimide labeled protein di lution method for the limiting cases; the analysis indicates that hemi fusion started at about 15 degrees C and increased over the range 20-2 5 degrees C. The initial rate of dequenching of the R-18 assay measure d within 2 min gives an accurate measure of membrane fusion above 30 d egrees C.