Abstract
Rabies virus entry into cultured hippocampal neurons was investigated by immunofluorescence and electron microscopy. Hippocampal neurons were susceptible to rabies virus infection and became filled with viral antigen 1 day after infection. Infection was inhibited by the lysosomotropic agents chloroquine and ammonium chloride. To study entry, neurons were adsorbed with rabies virus at 4°C and warmed to 37°C for short periods of time prior to fixation and localization of viral antigen by immunofluorescence microscopy. By 5 min at 37°C, viral antigen was localized to puncta in the cell body and dendrites and in synapses along dendrites. Little viral antigen was present in axons. Cells adsorbed with rabies virus were incubated with tracers for early endosomes. The endocytic tracers or markers Lucifer Yellow, transferrin receptor, dextran, and wheat germ agglutinin co-localized with rabies virus, indicating that rabies virus enters an endosome compartment shortly after uptake. Rabies virus also co-localized with LysoTracker Red, an acidotropic probe, indicating that some of the virus-containing endosomes are acidified. Rabies virus also co-localized with synapsin I, a synaptic vesicle marker, in nerve terminals but did not co-localize with lysosomal glycoprotein. By electron microscopy, after adsorption of virus and warming for 10 min, virus particles were present in coated pits, coated vesicles, and vacuolar membrane compartments in processes and axon terminals. It is concluded that rabies virus enters the somatodendritic domain and axon terminals of cultured hippocampal neurons by adsorptive endocytosis and is located in endosomes shortly after uptake.
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Adams, C. E. & Freedman, R. (1997) Nicotinic antagonist α-bungarotoxin binding to rat hippocampal neurons containing nitric oxide synthase. Brain Research 776, 111–6.
Alkondon, M., Reinhardt, S., Lobron, C., Hermsen, B., Maelicke, A. & Albuquerque, E. X. (1994) Diversity of nicotinic aceylcholine receptors in rat hippocampal neurons. II. The rundown and inward rectification of agonist-elicited whole-cell currents and identification of receptor subunits by in situ hybridization. Journal of Pharmacology and Experimental Therapeutics 271, 494–506.
Baer, G. M. & Lentz, T. L. (1991) Rabies pathogenesis to the central nervous system. In: The Natural History of Rabies (edited by Baer, G.M. ), pp. 105–20. Boca Raton: CRC Press.
Banker, G.A. & Cowan, W. M. (1977) Rat hippocampal neurons in dispersed cell culture. Brain Research 126, 397–425.
Barrantes, G. E., Westwick, J. & Wonnacott, S. (1994) Nicotinic acetylcholine receptors in primary cultures of hippocampal neurons: pharmacology and Ca+ permeability. Biochemical Society Transactions 22, 294S.
Bartlett, W. P. & Banker, G. A. (1984) An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture. I. Cells which develop without intercellular contacts. Journal of Neuroscience 4, 1944–53.
CÁceres, A., Banker, G. A. & Binder, L. (1986) Immunocytochemical localization of tubulin and microtubule-associated protein 2 during the development of hippocampal neurons in culture. Journal of Neuroscience 6, 714–22.
Cameron, P. L., SÑdhof, T. C., Jahn, R. & de Camilli, P. (1991) Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis. Journal of Cell Biology 115, 151–64.
Card, J. P., Rinaman, L., Lynn, R. B., Lee, B.-H., Meade, R. P., Miselis, R. R. & Enquist, L. W. (1993) Pseudorabies virus infection of the rat central nervous system: ultrastructural characterization of viral replication, transport, and pathogenesis. Journal of Neuroscience 13, 2525–39.
Charlton, K. M. & Casey, G. A. (1979) Experimental rabies in skunks. Immunofluorescence light and electron microscopic studies. Laboratory Investigation 41, 36–44.
de Camilli, P. & Takei, K. (1996) Molecular mechanisms in synaptic vesicle endocytosis and recycling. Neuron 16, 481–6.
de Hoop, M., von Poser, C., Lange, C., Ikonen, E., Hunziker, W., & Dotti, C. G. (1995) Intracellular routing of wild-type and mutated polymeric immunoglobulin receptor in hippocampal neurons in culture. Journal of Cell Biology 130, 1447–59.
Dotti, C. G., Sullivan, C. A. & Banker, G. A. (1988) The establishment of polarity by hippocampal neurons in culture. Journal of Neuroscience 8, 1454–68.
Fletcher, T.L., Cameron, P., de Camilli, P. & Banker, G. (1991) The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture. Journal of Neuroscience 11, 1617–26.
Fletcher, T. L., de Camilli, P. & Banker, G. (1994) Synaptogenesis in hippocampal cultures: evidence indicating that axons and dendrites become competent to form synapses at different stages of neuronal development. Journal of Neuroscience 14, 6695–706.
Gaudin, Y., Tuffereau, C., Segretain, D., Knossow, M. & Flamand, A. (1991) Reversible conformational changes and fusion activity of rabies virus glycoprotein. Journal of Virology 65, 4853–9.
Iwasaki, Y. (1991) Spread of virus within the central nervous system. In: The Natural History of Rabies (edited by Baer, G.M.), pp. 121–32. Boca Raton: CRC Press.
Iwasaki, Y. & Clark, H. F. (1975) Cell to cell transmission of virus in the central nervous system II. Experimental rabies in mouse. Laboratory Investigation 33, 391–9.
Iwasaki, Y., Liu, D.-S., Yamamoto, T. & Konno, H. (1985) On the replication and spread of rabies virus in the human central nervous system. Journal of Neuropathology and Experimental Neurology 44, 185–95.
Johnson, R. T. (1965) Experimental rabies. Studies of cellular vulnerability and pathogenesis using fluorescent antibody staining. Journal of Neuropathology and Experimental Neurology 24, 662–74.
Lentz, T. L. (1994) Molecular interaction of viruses with host cell receptors. In: Adhesion Molecules (edited by Wegner, C.D.), pp 223–51. London: Academic Press.
Lentz, T. L., Burrage, T. G., Smith, A. L., Crick, J. & Tignor, G. H. (1982) Is the acetylcholine receptor a rabies virus receptor? Science 215, 182–4.
Lentz, T.L., Chester, J., Benson, R. J. J., Hawrot, E., Tignor, G. H. & Smith, A. L. (1985) Rabies virus binding to cellular membranes measured by enzyme immunoassay. Muscle & Nerve 8, 336–45.
Lentz, T. L., Fu, Y. & Lewis, P. (1997) Rabies virus infection of IMR-32 human neuroblastoma cells and effect of neurochemical and other agents. Antiviral Research 35, 29–39.
Lycke, E., Hamark, B., Johansson, M., Krotochwil, A., Lycke, J. & Svennerholm, B. (1988) Herpes simplex virus infection of the human sensory neuron. An electron microscopy study. Archives of Virology 101, 87–104.
Marchand, C. F. & Schwab, M. E. (1986) Binding, uptake and retrograde axonal transport of herpes virus suis in sympathetic neurons. Brain Research 383, 262–70.
Marsh, M. & Helenius, A. (1989) Virus entry into animal cells. Advances in Virus Research 36, 107–51.
Mifune, K., Ohuchi, M. & Mannen, K. (1982) Hemolysis and cell fusion by rhabdoviruses. FEBS Letters 137, 293–7.
Mundigl, O., Matteoli, M., Daniell, L., Thomas-Reetz, A., Metcalf, A., Jahn, R. & de Camilli, P. (1993) Synaptic vesicle proteins and early endosomes in cultured hippocampal neurons: differential effects of Brefeldin A in axons and dendrites. Journal of Cell Biology 122, 1207–21.
Murphy, F. A. (1977) Rabies pathogenesis. Brief review. Archives of Virology 54, 279–97.
Murphy, F. A., Harrison, A. K., Winn, W. C. & Bauer, S. P. (1973) Comparative pathogenesis of rabies and rabies-like viruses. Infection of the central nervous system and centrifugal spread of virus to peripheral tissues. Laboratory Investigation 29, 1–16.
Parton, R. G., Simons, K. & Dotti, C. G. (1992) Axonal and dendritic endocytic pathways in cultured neurons. Journal of Cell Biology 119, 123–37.
Paulson, J. C. (1985) Interactions of animal viruses with cell surface receptors. In: The Receptors (edited by Conn, P.M.), pp. 131–219. Orlando: Academic Press.
Penfold, M. E. T., Armati, P. & Cunningham, A. L. (1994) Axonal transport of herpes simplex virions to epidermal cells: evidence for a specialized mode of virus transport and assembly. Proceedings of the National Academy of Sciences USA 91, 6529–33.
Perrin, P., Portnoï, D. & Sureau, P. (1982) Étude de l’adsorption et de la pénétration du virus rabique: interactions avec les cellules BHK21 et des membranes artificielles. Annales de Virologie (Institut Pasteur) 133E, 403–22.
Sodiek, B., Ebersold, M. W. & Helenius, A. (1997) Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. Journal of Cell Biology 136, 1007–21.
Superti F., Derer M., & Tsiang, H. (1984) Mechanism of rabies virus entry in CER cells. Journal of General Virology 65, 781–9.
Tsiang, H. & Superti, F. (1984) Ammonium chloride and chloroquine inhibit rabies virus infection in neuroblastoma cells. Archives of Virology 81, 377–82.
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Lewis, P., Lentz, T.L. Rabies virus entry into cultured rat hippo campalneurons. J Neurocytol 27, 559–573 (1998). https://doi.org/10.1023/A:1006912610044
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DOI: https://doi.org/10.1023/A:1006912610044