Rabies virus entry at the neuromuscular junction in nerve-muscle cocultures

Early events in rabies virus entry into neurons were investigated in chick spinal cord-muscle cocultures. Rabies virus (CVS strain) was adsorbed to th e surface of cells in the cold. At times up to 10 min of warming to 37 degr ees C, virus was most intensely localized to dense swellings on the myotube surface. Texas Red-labeled alpha-bungarotoxin, which binds to nicotinic ac etylcholine receptors, colocalized precisely with virus at the densities id entifying these regions as neuromuscular junctions. Rabies virus also coloc alized in the junctions with synapsin I, a marker for synaptic vesicles. Th e endosome tracers Lucifer Yellow, Texan Red-dextran, and rhodamine-wheat g erm agglutinin were added to the cultures at the end of the virus adsorptio n period and the cultures were warmed. At 10 min, rabies virus and tracers colocalized at neuromuscular junctions and nerve terminals. At 30 min, rabi es virus and tracers showed more intense fluorescence over nerve fibers and nerve cell bodies. At 60 min, nerve terminals, nerve fibers, and nerve cel l bodies showed intense fluorescence and colocalization for rabies virus an d tracers. LysoTracker Red, a marker for acidic compartments, colocalized w ith rabies virus at nerve-muscle contacts. These findings show that in nerv e-muscle cocultures, the neuromuscular junction is the major site of entry into neurons. Colocalization of virus and endosome tracers within nerve ter minals indicates that virus resides in an early endosome compartment, some of which are acidified. The progressive increase of virus and tracers in ne rve fibers and nerve cell bodies over time is consistent with retrograde tr ansport of endocytosed virus from the motor nerve terminal. (C) 2000 John W iley & Sons, Inc.