During rabies virus infection in vivo, the virus gains entry into neurons at peripheral nerve terminal traverses the neuron, replicates, and is transferred transynaptically. Thus, this virus represents an ideal marker to study the trafficking and targeting of membrane compartments containing virus throughout the neuron. This project is designed to study the intracellular pathways taken by rabies virus during infection of neurons. Using rabies virus as a probe, the function, transport, and targeting of membrane compartments, especially endosomes, in neurons will be investigated. These processes will be studied in a model in vitro system in which neurons are cultured in the inner chamber of a multichambered culture system. Neurites from these neurons cross a fluid-impermeable barrier and innervate muscle cells and other neurons in an outer chamber. This system allows nerve terminals and neuromuscular junctions to be exposed to virus in a manner similar to that occurring in vivo. The subsequent uptake of virus by nerve terminals and movement of virus and associated membrane compartments in the neuron will be monitored by double immunofluorescence and high resolution confocal scanning microscopy and video microscopy using fluorescently labeled antibodies or fluorescent markers for virus, endosomes, synaptic vesicles, and lysosomes. Virus or viral glycoprotein will also be traced by electron microscopy, immunocytochemistry, and cell fractionation. It will be determined whether virus enters nerve terminals by fusion or by receptor-mediated endocytosis and whether it is incorporated into an endosome compartment. Endosomes in nerve terminals will be identified using endocytic tracers and free flow electrophoresis. The effect of synaptic vesicle release on uptake of virus particles and the relationship of endosomes to recycling synaptic vesicle membrane will be determined. It will be noted whether acidification of endosomes and uncoating of virus takes place in the nerve terminals or cell body. The transport and destination of virus and viral coat glycoprotein daring viral replication will be studied to gain information on polarized targeting of newly synthesized proteins. Retrograde and anterograde transfer of virus across synapses will be investigated to study mechanisms of transynaptic transfer. The possibility that rabies virus uses neuronal nicotinic acetylcholine receptors as host cell receptors will be investigated. Finally, the in vitro system will be used to assay the potential effectiveness of antiviral agents at different stages in pathogenesis. Agents to be tested include lysosomotropic agents, anti-rabies antibodies, and synthetic peptides of the viral glycoprotein and the acetylcholine receptor. These studies will provide new information on the poorly understood processes of endocytosis, endosome function, and membrane trafficking in neurons. In addition, they will provide information on the pathogenesis of rabies and on possible sites for intervention at the attachment stage of the infectious cycle that may be applicable to other neurotropic viruses.