FUSION BETWEEN NEWCASTLE-DISEASE VIRUS AND ERYTHROCYTE-GHOSTS USING OCTADECYL RHODAMINE-B FLUORESCENCE ASSAY PRODUCES DEQUENCHING CURVES THAT FIT THE SUM OF 2 EXPONENTIALS

The kinetics of fusion between Newcastle disease virus and erythrocyte ghosts has been investigated with the octadecyl Rhodamine B chloride assay [Hoekstra, De Beer, Klappe, and Wilschut (1984) Biochemistry 23, 5675-5681], and the data from the dequenching curves were fitted by n on-linear regression to currently used kinetic models. We used direct computer-assisted fitting of the dequenching curves to the mathematica l equations. Discrimination between models was performed by statistica l analysis of different fits. The experimental data fit the exponentia l model previously published [Nir, Klappe, and Hoekstra (1986) Biochem istry 25, 2155-2161] but we describe for the first time that the best fit was achieved for the sum of two exponential terms: A(1)[1-exp(-k(1 )t)]+A(2)[1-exp(-k(2)t)]. The first exponential term represents a fast reaction and the second a slow dequenching reaction. These findings r eveal the existence of two independent, but simultaneous, processes du ring the fusion assay. In order to challenge the model and to understa nd the meaning of both equations, 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 disulp hide-reducing agents or inhibitors of viral neuraminidase activity), a nd the same computer fitting scheme was followed. The first exponentia l equation represents the viral protein-dependent fusion process itsel f, because it is affected by the assay conditions. The second exponent ial equation accounts for a nonspecific reaction, because it is comple tely independent of the assay conditions and hence of the viral protei ns. An interpretation of this second process is discussed in terms of probe transfer between vesicles.