Kinetics of virus adsorption to single cells using fluorescent membrane probes and multiparameter flow cytometry

Different genetic stains of avian RNA tumor virus (ATV) were labeled with the fluorescent membrane probe R-18 (rhodamine conjugated to a hydrocarbon chain) and cellular receptors for virus infection were analyzed on a rapid, single-cell basis by a multiparameter cell sorter. Chicken cells genetically susceptible to various R-18 ATV were found to adsorb much more virus, as measured by increased fluorescent binding, than did genetically resistant chicken cells. Virus binding to receptor sites could be saturated with increased concentrations of labeled virus. This binding could be altered by removal of the polycation, polybrene, indicating the important influence of electrostatic forces. Correlated time measurements of virus binding to single-cells were taken with these fluorescence measurements allowing for a minute-to-minute study of the kinetics of viral adsorption to resistant and susceptible cells. The ratio of fluorescence (proportional to the number of virions bound per cell) to light scatter (proportional to cell surface area) on a cell-to-cell basis was analyzed to examine the heterogeneity in fluorescent virion bound per unit cell surface area within a given cell type. With these calculations, it was found that a large amount, but not all, of observed fluorescence heterogeneity merely reflects differences in cell surface areas. However, there are significant differences in viral receptor site densities within this supposedly homogeneous population of cells. This study represents a successful application of fluorescent membrane probes and flow cytometry to the study of cellular responses to viral infection at the single-cell level. Since large numbers of cells can be examined rapidly, small subpopulations of live virally susceptible or resistant cells can be cloned by multiparameter cell sorting.