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 (tlag) following pH reduction. Whereas tlag represents only a subpopulation of fusing viruses with the shortest delay time we suggest here that a representative mean lag time mu lag of virus-cell fusion can be deduced from the R 18-assay. Kinetics of FDQ reflects the cumulative distribution function of lag times tau lag of single fusion events with the mean value mu lag. We show that tau 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, taulag and mu 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 mu 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.
Ludwig
K
K
Mathematisch-Naturwissenschaftliche Fakultät I, Humboldt-Universität zu Berlin, Germany.
Korte
T
T
Herrmann
A
A
D002478:Cells, Cultured; D004910:Erythrocyte Membrane / Q000378:metabolism* / Q000821:virology*; D005453:Fluorescence; D006389:Hemagglutinins, Viral / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000254:growth & development / Q000378:metabolism*; D007700:Kinetics; D013696:Temperature
D006389:Hemagglutinins, Viral