Fusion of fluorescently labeled Sendai virus envelopes with living cultured cells as monitored by fluorescence dequenching.
Fluorescently labeled (bearing N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine (N-NBD-PE)) reconstituted Sendai virus envelopes (RSVE) were used to study fusion between the viral envelopes and cultured living cells such as lymphoma, Friend erythroleukemia cells (FELC) and L cells. Incubation of fusogenic viruses with the above cell lines resulted in a relatively high degree (40-45%) of fluorescence dequenching. On the other hand, incubation of unfusogenic (trypsin or phenylmethylsulfonylfluoride (PMSF)-treated) RSVE with these cells led to very little (6-9%) fluorescence dequenching. The degree of fluorescence dequenching was linearly correlated to the surface density of the virus-inserted N-NBD-PE molecules. Fluorescence photobleaching recovery experiments showed that fusion of fluorescent RSVE with FELC resulted in an infinite dilution of the fluorescent molecules in the recipient cell membranes. The fluorescent probe 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (N-NBD-Cl) was covalently attached to envelopes of intact Sendai virions without significantly impairing their biological activity. Incubation of fluorescently labeled, intact Sendai virions with cultured cells resulted in about 20% fluorescence dequenching. The present data clearly indicate that fluorescently labeled Sendai virions can be used for a quantitative estimation of the degree of virus-membrane fusion.
D000818:Animals; D002459:Cell Fusion; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D006801:Humans; D007739:L Cells; D004915:Leukemia, Erythroblastic, Acute; D008223:Lymphoma; D051379:Mice; D010222:Parainfluenza Virus 1, Human / Q000502:physiology* / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes