Inactivation of PR8 influenza virus through the octadecylrhodamine B chloride membrane marker.

Abstract

The octadecylrhodamine B chloride (R18) membrane marker was incorporated into PR8 influenza viruses and virus receptor (GD1a-) containing small unilamellar vesicles (SUV). Both were tested in a fusion/lipid transfer assay [Wunderli-Allenspach, H., & Ott, S. (1990) Biochemistry 29, 1990-1997] to find out whether incorporation into artificial and biological membranes yields equivalent results. The R18 assay is based on incorporation of quenched concentrations of the label into donor membranes and monitoring of the dequenching upon its dilution into unlabeled acceptors. With PR8 viruses and R18-labeled SUV, a fast, hemagglutinin-specific fusion takes place at pH 5.3, independently of the initial quenching. At neutral pH, a slow, nonspecific R18 transfer occurs. Both processes follow second-order kinetics. Upon incubation of R18-labeled PR8 viruses with unlabeled SUV or LUV in neutral buffer, transfer is also found. At pH 5.3, a complex dequenching curve best described with superposition of two second-order functions was encountered: a fast, hemagglutinin-specific component and a slow, nonspecific component. A decreasing proportion of the fast fusion was found with increasing initial quenching of labeled virus. Extrapolation showed that full fusion activity is obtained only with low initial quenching (20-30%). The gradual inactivation of the virus by increasing amounts of R18 was confirmed with biological assays (e.g., infectivity). The R18 surface density is much higher in viruses than in liposomes to obtain the same initial quenching. Analysis with the Stern-Volmer plot revealed that R18 monomers and dimers contribute to the quenching in labeled PR8 viruses, whereas only dimers determine the quench curve in liposomes.