A common mechanism for influenza virus fusion activity and inactivation

The fusion of influenza virus (A/PR/8/34 strain) with PC- 12 cells was monitored by a fluorescence assay, and the results were analyzed with a mass-action model which could explain and predict the kinetics of fusion. The model accounted explicitly for the reduction in the fusion rate constant upon exposure of the virus to low pH, either for the virus alone in suspension or for the virus bound to the cells. When the pH was lowered without previous viral attachment to cells, an optimal fusion activity was detected at pH 5.2. When the virus was prebound to the cells, however, reduction of pH below 5.2 resulted in enhanced fusion activity at the initial stages. These results were explained by the fact that the rate constants of both fusion and inactivation increased severalfold at pH 4.5 or 4, compared to those at pH 5.2. At pH 5.2, lowering the temperature from 37 to 20 or 4 "C resulted in a decrease in the fusion rate constant by more than 30- or 1000-fold, respectively. Inactivation of the virus when preincubated in the absence of target membranes at pH 5 was found to be rapid and extensive at 37 OC, but was also detected at 0 OC. Our results indicate a strong correlation between fusion and inactivation rate constants, suggesting that the rate-limiting step in viral hemagglutinin (HA)-mediated fusion, that is, rearrangement of viral glycoproteins at the contact points with the target membrane, is similar to that involved in fusion inactivation

Interactions of Influenza Virus with Cultured Cells: Detailed Kinetic Modeling of Binding and Endocytosis

We performed a detailed kinetic analysis of the uptake of influenza virus (A/PR8/34) by Madin Darby canine kidney (MDCK) cells in culture. Experimental procedures were based on the relief of fluorescence self-quenching of the fluorescent probe octadecylrhodamine B chloride (R18) incorporated in the viral envelope. Equilibrium for binding of influenza virus to MDCK cells (2.5 × 106/mL) was reached quicker with temperature increases due to a faster dynamic mobility of the particles. We deduced that there are two kinds of binding sites for influenza virus in MDCK cells and determined the kinetic parameters of the binding process (adhesion and detachment rate constants), using a mass action kinetic model. As the temperature increases, the number of binding sites for influenza virus decreases, especially the high-affinity binding sites, whereas the value of the affinity constant for virus binding to the binding site, k, increases. Nevertheless, the binding association constant at equilibrium Ki, which is given by Ki = Niki, where Ni is the number of binding sites per cell, declines as the temperature increases. When endocytosis occurs, the total uptake of virions by the cells is larger than that observed in the process of binding at the same temperature, and the uptake proceeds for longer times. Using our mass kinetic model, we determined that at 20 °C, the rate constant of endocytosis, ε, for influenza virus with this cell line is 2.6 × 10-4 s-1, i.e., in the same range as in studies on endocytosis of liposomes