Kinetics of fusion with cells of reconstituted Sendai virus envelopes lacking hemagglutinin-neuraminidase

Abstract

The fusion potential of reconstituted Sendai virus envelopes containing only the F protein (F-virosomes) has been assessed. F-virosomes and F, HN-virosomes were prepared by solubilization of the intact virus in Triton X-100 followed by its removal using SM-2 biobeads. Viral envelopes containing HN whose disulphide bonds were irreversibly reduced (HNred) were also prepared by treating the envelopes with dithiothreitol followed by dialysis. Both F-virosomes and F,HNred-virosomes hemolysed red blood cells in the presence of wheat germ agglutinin. The rates and extent of hemolysis induced by these virosomes were, however, significantly lower than that induced by F,HN-virosomes. Using a fluorescence probe based membrane mixing fusion assay, F- and F,HNred-virosomes were found to fuse with cultured HeLa cells in the presence of wheat-germ agglutinin. A direct comparison of the fusion activity of F,HN-virosomes and F-virosomes was made by using desialylated HepG2 cells as target containing the asialoglycoprotein receptor (ASGP-R) that binds to a terminal beta-galactose moiety of F protein. A 2- to 3-fold enhancement in the fusion rate when HN was included in the viral envelope was observed. Based on the kinetic data, a model for fusion of paramyxo-virosomes with HepG2 cells is proposed.