J. gen. Virol. (1984), 65, 781-789. Printed in Great Britain Key words: rabies virus~entry~adsorptiveendocytosis 781 Mechanism of Rabies Virus Entry into CER Cells By F. SUPERTI, M. DERER 1 AND H. TSIANG* UnitO de Recherche sur la Rage, Institut Pasteur, 25 Rue du Dr Roux, 75 724, Paris Cddex 15, France and 1Laboratoire de Cytologie, Universitb P. et M. Curie, Paris et Unitd INSERM 153, Paris, France (Accepted 29 December 1983) SUMMARY The early steps of rabies virus (CVS) infection in vitro were studied in chicken embryo-related (CER) cells. The infection was monitored by looking for specific intra- cytoplasmic viral inclusions using anti-rabies fluorescein isothiocyanate at 24 h after the addition of virus. The attachment of rabies virus to CER cells was shown to be inhibited by pretreatment of the cells with neuraminidase. These cells recovered their susceptibility to rabies virus infection 6 h after removal of the enzyme. Treatment of CER cells with neuraminidase after the viral attachment step did not inhibit infection. The subsequent delivery of infectious virions into acid prelysosomal vacuoles or lysosomes was studied using lysosomotropic agents. Ammonium chloride and chloroquine were used to prevent the virus fusion step thus preventing infection. Both drugs were shown to inhibit the early steps of infection, NH4C1 having a much earlier effect than chloroquine. The two drugs had no effect on the attachment step nor did NHAC1 inhibit virus multiplication. The use of metabolic inhibitors (2-deoxy-D-glucose and sodium azide) shows that the entry of rabies virus into CER cells does not require the involvement of cellular energy processes. In electron microscopy studies, the presence of rabies virus particles was detected in coated pits and coated vesicles as well as in uncoated vesicles, and later in lysosomes. These data indicate that the mechanism by which rabies virus enters CER cells is probably through adsorptive endocytosis and does not require the participation of cellular metabolic active processes. INTRODUCTION Rabies virus is known to be a neurotropic virus from examination of infected tissues in vivo (Matsumoto, 1962; Murphy, 1977). Results of experiments in vitro have also confirmed the neurotropic aspect of rabies virus infection of dissociated primary cultures of ceils derived from neural tissues (Tsiang et aL, 1983). It has also been reported that before entering the nervous system, rabies virus infects striated muscles at the site of virus inoculation (Murphy & Bauer, 1974; Harrison & Murphy, 1978). Experimental evidence has shown that in the next step, rabies virus is probably taken up into the peripheral nervous system at the neuromuscular junction (Watson et al., 1981), and that it reaches the central nervous system by passive transport through the retrograde axoplasmic flow (Bijlenga & Heaney, 1978; Tsiang, 1979). Furthermore, some experiments suggest that the nicotinic receptor for acetyl choline is also a receptor for rabies virus (Lentz et al., 1982). On the other hand, little is known about the entry pathway of rabies virus into susceptible culture cells. Most of the enveloped viruses have been shown to enter the host cells by adsorptive endocytosis (Dales &Pons, 1976; Talbot & Vance, 1980; Helenius et al., 1980; Marsh & Helenius, 1980; Matlin et al., 1981, 1982; Schlegel et al., 1982a). The paramyxoviruses, however, enter the cells by a different mechanism; these viruses are characterized by their ability to fuse with the cellular membrane, although this property varies among different strains (Lenard & Miller, 1982). 0022-1317/84/0000-5877$02.00 © 1984 SGM