Fusion between Newcastle disease virus and erythrocyte ghosts using octadecyl Rhodamine B fluorescence assay produces dequenching curves that fit the sum of two exponentials.
The kinetics of fusion between Newcastle disease virus and erythrocyte ghosts has been investigated with the octadecyl Rhodamine B chloride assay [Hoekstra, De Boer, Klappe, and Wilschut (1984) Biochemistry 23, 5675-5681], and the data from the dequenching curves were fitted by non-linear regression to currently used kinetic models. We used direct computer-assisted fitting of the dequenching curves to the mathematical equations. Discrimination between models was performed by statistical analysis of different fits. The experimental data fit the exponential model previously published [Nir, Klappe, and Hoekstra (1986) Biochemistry 25, 2155-2161] but we describe for the first time that the best fit was achieved for the sum of two exponential terms: A1[1-exp(-k1t)]+A2[1-exp(-k2t)]. The first exponential term represents a fast reaction and the second a slow dequenching reaction. These findings reveal the existence of two independent, but simultaneous, processes during the fusion assay. In order to challenge the model and to understand the meaning of both equation, fusion experiments were carried out under different conditions well known to affect viral fusion (changes in pH, temperature and ghost concentration, and the presence of disulphide-reducing agents or inhibitors of viral neuraminidase activity), and the same computer fitting scheme was followed. The first exponential equation represents the viral protein-dependent fusion process itself, because it is affected by the assay conditions. The second exponential equation accounts for a nonspecific reaction, because it is completely independent of the assay conditions and hence of the viral proteins. An interpretation of this second process is discussed in terms of probe transfer between vesicles.
Cobaleda
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C
Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Salamanca, Spain.
García-Sastre
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A
Villar
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E
D000818:Animals; D002642:Chick Embryo; D004910:Erythrocyte Membrane / Q000382:microbiology*; D005456:Fluorescent Dyes; D007700:Kinetics; D008561:Membrane Fusion; D009522:Newcastle disease virus / Q000502:physiology*; D012235:Rhodamines; D013050:Spectrometry, Fluorescence; D013552:Swine
D005456:Fluorescent Dyes; D012235:Rhodamines; C044542:octadecyl Rhodamine B chloride