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. Experimenta l 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 x 10(6)/mL) was reached quicker with temperature increases due to a fast er dynamic mobility of the particles. We deduced that there are two kinds o f binding sites for influenza virus in MDCK cells and determined the kineti c parameters of the binding process (adhesion and detachment rate constants ), using a mass action kinetic model. As the temperature increases, the num ber of binding sites for influenza virus decreases, especially the high-aff inity binding sites, whereas the value of the affinity constant for virus b inding to the binding site, k, increases. Nevertheless, the binding associa tion constant at equilibrium K-i, which is given by K-i = N(i)k(i), where N -i is the number of binding sites per cell, declines as the temperature inc reases. When endocytosis occurs, the total uptake of virions by the cells i s larger than that observed in the process of binding at the same temperatu re, and the uptake proceeds for longer times. Using our mass kinetic model, we determined that at 20 degrees C, the rate constant of endocytosis, epsi lon, for influenza virus with this cell line is 2.6 x 10(-4) s(-1), i.e., i n the same range as in studies on endocytosis of liposomes.