Analysis of delay times of hemagglutinin-mediated fusion between influenza virus and cell membranes

We have studied the kinetics of low pH-induced fusion between influenza virus A/PR 8/34 and human erythrocyte membranes in suspension by using an assay based on fluorescence dequenching (FDQ) of the lipophilic dye octadecylrhodamine B chloride (R 18). As shown previously (Clague et al. 1991) the onset of FDQ is preceded by a characteristic lag time (t_lag) following pH reduction. Whereas t_lag represents only a subpopulation of fusing viruses with the shortest delay time we suggest here that a representative mean lag time µ_1ag of virus-cell fusion can be deduced from the R 18-assay. Kinetics of FDQ reflects the cumulative distribution function of lag times _lag of single fusion events with the mean value µ_lag. We show that t_lag obtained from the onset of FDQ does not always reflect the fusion behaviour of the whole population of fusing viruses. While both lag times, t_lag and µ_lag exhibit a similar temperature dependence we found a significantly different dependence of both delay times on virus inactivation by low pH-pretreatment. We conclude that the mean lag time µ_lag appears to be a more appropriate parameter describing the kinetics of virus-cell fusion. The analysis of delay times offers a new approach to test the validity of different kinetic models of HA-mediated fusion and to gain valuable information about HA-mediated fusion. The analysis confirms that the inactivation process proceeds via steps of the formation of the fusion pore. Although the increase of lag times can be explained by a depletion of fusion competent HA's, our data suggest that intermediate structures of HA along the inactivation pathway can still transform into a fusion site.Abbreviations FDQ fluorescence dequenching - HA hemagglutinin - PBS phosphate buffered saline - R18 octadecylrhodamine B chloride - t_lag lag time - µ_lag mean lag time - _lag individual delay timeCorrespondence to: A. Herrmann