Fluorescence Photobleaching Recovery as a Method To Quantitate Viral Envelope-Cell Fusion: Application To Study Fusion of Sendai Virus Envelopes with Cells

A method to quantitate viral envelope-cell fusion at the single-cell level is presented. The method is based on the incorporation of nonquenching concentrations of a fluorescent lipid probe into the viral envelope; fluorescence photobleaching recovery (FPR) is then applied to measure the lateral mobilization of the probe in the cell membrane following fusion. In adsorbed (unfused) viral envelopes, the probe is constricted to the envelope and is laterally immobile on the micrometer scale of FPR. After fusion, the envelope lipids intermix with the plasma membrane, the probe becomes laterally mobile, and the fraction of fused viral envelopes can be extracted from the fraction of mobile probe molecules. The method has several advantages: (i) It clearly distinguishes fused from internalized envelopes, as probes in the latter are immobile in FPR studies; (ii) focusing the laser beam on specific regions of the cell enables region-specific measurements of the fusion level; (iii) one cell is measured at a time, enabling studies on the distribution of the fusion level within the cell population. The new method was employed to study fusion of reconstituted Sendai virus envelopes (RSVE) containing N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine with several cell types. Experiments with human erythrocytes demonstrated that the lateral mobilization measured is due to fusion and not the result of exchange processes. The extent of RSVE-erythrocyte fusion determined by FPR was similar to that measured by two other independent methods (fluorescence dequenching and removal of adsorbed RSVE by dithiothreitol). FPR experiments on other cell types (BHK-21, HeLa, GM-22 rat hepatoma cells, and mouse spleen lymphocytes), which are active in endocytosis, showed different degrees of RSVE-cell fusion depending on the cell region examined. There were also cell type dependent differences in the distribution of RSVE-cell fusion within the cell population. These results are discussed in light of the scope of the FPR method for studies on viral envelope-cell fusion at the single-cell level.