Descrição
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. http://dx.doi.org/10.1021/bi9812524
; Ramalho-Santos, João 
