Fusion of native Sendai virions with human erythrocytes : Quantitation by fluorescence photobleaching recovery |
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| Authors: | Benjamin Aroeti Yoav I Henis |
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| Institution: | 1. Faculty of Engineering, Utsunomiya University, Utsunomiya, Tochigi, 321-8585, Japan;2. Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi, 980-5877, Japan;3. Department of Biomolecular Chemistry, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-8522, Japan;1. Department of Psychiatry, Otto-von-Guericke University, Faculty of Medicine, Magdeburg, Germany;2. Institute of Biochemistry and Cell Biology, Otto-von-Guericke University, Faculty of Medicine, Magdeburg, Germany;3. Leibniz Institute for Neurobiology, Magdeburg, Germany;1. Department of Chemical Engineering, School of Chemical and Petroleum Engineering, Shiraz University, Shiraz, Iran;2. Natural Gas Engineering Department, School of Chemical and Petroleum Engineering, Shiraz University, Shiraz, Iran;3. Institut de Recherche en Génie Chimique et Pétrolier (IRGCP), Paris Cedex, France;4. Department of Chemical and Petroleum Engineering, Schulich School of Engineering, University of Calgary, Calgary, AB T2N 1N4, Canada;5. Discipline of Chemical Engineering, School of Engineering, University of KwaZulu-Natal, Howard College Campus, King George V Avenue, Durban, 4041, South Africa;1. Center for International Research on Integrative Biomedical Systems (CIBiS), Institute of Industrial Science (IIS), The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan;2. Department of Mechano-Informatics, Graduate School of Information Science and Technology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan |
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| Abstract: | We have recently developed a method to quantitate the fusion of reconstituted viral envelopes with cells by fluorescence photobleaching recovery (FPR) (Aroeti, B & Henis, Y I, Biochemistry 25 (1986) 4588). The method is based on the incorporation of non quenching concentrations of the fluorescent lipid probe N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)phosphatidylethanolamine during the reconstitution of the viral envelopes (the latter probe does not incorporate efficiently into the membrane of native virions). In the present work, we employed the fluorescent dye octadecyl rhodamine B chloride (R18), which can be incorporated directly into the membrane of native enveloped virions, to extend the FPR method to study fusion between native Sendai virions and intact human erythrocytes. The R18 fluorescence was found to be quenched in the viral envelope at the concentration range required for the FPR experiments, possibly due to preferential insertion of the probe into specific domains in the viral membrane. We therefore developed a correction (presented in the Appendix) which takes into account the lower quantum yield of the probe molecules in the membranes of unfused virions in the calculation of the fraction of fused virions from the FPR experiments. The results demonstrate that the method does indeed measure virus-cell fusion, and that the contribution of exchange to the measurements is not significant. The applicability of the method was further verified by the similarity of the results to those obtained independently by fluorescence dequenching measurements, and its ability to measure the distribution of virus-cell fusion within the cell population was demonstrated. These results suggest that the use of R18 can enlarge the scope of the FPR experiments to study the fusion of native virions with cells. |
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