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.

P74 Mediates Specific Binding of Autographa californica M Nucleopolyhedrovirus Occlusion-Derived Virus to Primary Cellular Targets in the Midgut Epithelia of Heliothis virescens Larvae

“…This level of reduced infectivity is consistent with infectivity losses reported for other R-18-labeled viruses (24). We next demonstrated that H. virescens midgut cells could become saturated with R-18-labeled ODV and that labeled ODV binding could be reduced progressively by the addition of increasing amounts of excess unlabeled ODV.…”

Influence of the Membrane Dye R18 and of DMSO on Cell Penetration of Guanidinium‐Rich Peptides

“…As a π ‐system with delocalized positive charge, R18 may act as a transporter of molecules with likewise delocalized anionic charges into and across phospholipid bilayers . Dramatic effects of R18 and other membrane markers have been observed in investigations of viral membrane fusion, all the way to inactivation of the PR8 influenza virus . Finally, fluorescent lipid probes have been shown to generate light‐induced artifacts under laser irradiation of confocal fluorescence microscopy experiments …”

“…[29] Dramatic effects of R18 and other membrane markers have been observed in investigations of viral membrane fusion, all the way to inactivation of the PR8 influenza virus. [30] Finally, fluorescent lipid probes have been shown to generate lightinduced artifacts under laser irradiation of confocal fluorescence microscopy experiments. [31 -34] In summary, and in agreement with our experimental result, we must state that these fluorescent dyes are by no means 'innocent' labels, and that 'caution is needed when using direct labeling of biological membranes'.…”

Respiratory Syncytial Virus-Neutralizing Monoclonal Antibodies Motavizumab and Palivizumab Inhibit Fusion

“…The lipid-mixing assay may indicate that the hairpin conformation, which is the point at which lipid mixing occurs, is achieved in the presence of these antibodies and that palivizumab and motavizumab block the transition from a hairpin conformation to a postfusion conformation. However, several reports suggest that R18 can passively transfer from virus to target cell by shear proximity of the membranes (5,36,46,54,55). Therefore, it is possible that R18 may transfer from virus to target cell upon the initial interaction of the F protein with the target cell membrane or the prehairpin conformation.…”