Kinetics of fusion and lipid transfer between virus receptor containing liposomes and influenza viruses as measured with the octadecylrhodamine B chloride assay.
Octadecylrhodamine B chloride (R18) and ganglioside GD1a (virus receptor) were incorporated into small unilamellar liposomes [Hoekstra et al. (1984) Biochemistry 23, 5675-5681]. Upon interaction of these liposomes with PR8 influenza viruses without prebinding, two types of dequenching were observed at 37 degrees C, both second-order processes: a fast reaction at pH 5.3, 2k = 17.53 x 10(-3) (Q.s)-1, and a slow reaction at pH 7.4, 2k = 0.335 x 10(-3) (Q.s)-1. The maximal level of dequenching was the same for both. Upon prebinding of liposomes to PR8 viruses (30 min, 0 degrees C, pH 7.4) at high concentrations, a very fast dequenching occurred when the prebinding mixture was diluted into prewarmed (37 degrees C) 10 mM PBS, pH 5.3. For the initial phase, a first-order rate constant of 0.5 s-1 could be extrapolated. After a quick drop in velocity during the first 30 s, the reaction was kinetically indistinguishable from the one found without prebinding. A second-order process with 2k = 16.52 x 10(-3) (Q.s)-1 became rate-limiting. The fast reactions at pH 5.3 can be abolished by inactivation or removal of the virus hemagglutinin. We conclude that the reaction at pH 5.3 reflects the hemagglutinin-dependent fusion process known to occur between influenza viruses and partner membranes at low pH; however, second-order kinetics indicate that specific binding rather than fusion is the rate-limiting step. For the slow dequenching, which is not affected by prebinding, the rate constant is 20 times lower than for the fast reaction, and the process is independent of viral hemagglutinin.(ABSTRACT TRUNCATED AT 250 WORDS)
Wunderli-Allenspach
H
H
Department of Pharmacy, Biopharmacy, Federal Institute of Technology, Zürich, Switzerland.
Ott
S
S
D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050356:Lipid Metabolism; D008081:Liposomes / Q000378:metabolism*; D008561:Membrane Fusion; D009975:Orthomyxoviridae / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism*; D012235:Rhodamines; D014966:Xanthenes
D008081:Liposomes; D011991:Receptors, Virus; D012235:Rhodamines; D014966:Xanthenes; C044542:octadecyl Rhodamine B chloride