Rabies virus (RV), a highly neurotropic enveloped virus, is known to spread within the CNS by means of axonal transport. Although the envelope spike glycoprotein (G) of cell-free virions is required for attachment to neuronal receptors and for virus entry, its necessity for transsynaptic spread remains controversial. In this work, a G gene-deficient recombinant RV (SAD ∆G) complemented phenotypically with RV G protein (SAD ∆GMG) has been used to demonstrate the absolute requirement for G in virus transfer from one neuron to another, both in vitro, in neuronal cell cultures (cell line and primary cultures), and in vivo, in murine animal models. By using a model of stereotaxic inoculation into the rat striatum, infection is shown to be restricted to initially infected cells and not transferred to secondary neurons. In mouse as in rat models of infection, the limited infection did not cause any detectable symptoms, suggesting that Gdeficient RV recombinants might be valuable as non-pathogenic, single-round vectors for expression of foreign genes.
SUMMARYThe 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 intracytoplasmic 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.
SUMMARYThe role of gangliosides in rabies virus infection of chick embryo-related (CER) cells was investigated. Cultured cells were pretreated with neuraminidase to render the cells transiently non-susceptible to viral infection. Incubation of these desialylated cells with gangliosides allowed them to incorporate exogenous gangliosides and they recovered their susceptibility to rabies virus infection. Infection of CER cells was monitored by specific fluorescence 24 h after virus inoculation. The use of individual purified gangliosides or mixtures of two gangliosides to restore cellular susceptibility to viral infection showed that GTlb and GQlb were the most effective. The disialogangliosides were also active, principally GD 1 b, whereas GM 1, GM3 were poorly active and GD3 inactive. Incubation of rabies virus with gangliosides prior to virus infection reduced the percentage of infected cells. The results indicate that highly sialylated gangliosides are part of the cellular membrane receptor structure for the attachment of infective rabies virus. However, it is possible that other glycoconjugates such as glycoproteins or glycolipids also participate as components of a receptor structure for rabies virus.
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