Rabies virus modifies host behaviour through a snake-toxin like region of its glycoprotein that inhibits neurotransmitter receptors in the CNS

Abstract

Rabies virus induces drastic behaviour modifications in infected hosts. The mechanisms used to achieve these changes in the host are not known. The main finding of this study is that a region in the rabies virus glycoprotein, with homologies to snake toxins, has the ability to alter behaviour in animals through inhibition of nicotinic acetylcholine receptors present in the central nervous system. This finding provides a novel aspect to virus receptor interaction and host manipulation by pathogens in general. The neurotoxin-like region of the rabies virus glycoprotein inhibited acetylcholine responses of α4β2 nicotinic receptors in vitro, as did full length ectodomain of the rabies virus glycoprotein. The same peptides significantly altered a nicotinic receptor induced behaviour in C. elegans and increased locomotor activity levels when injected into the central nervous system of mice. These results provide a mechanistic explanation for the behavioural changes in hosts infected by rabies virus.

Figure 1

Neurotoxin-like peptides of the rabies virus glycoprotein contain polymorphic and highly conserved residues (a) and differ in binding to homologue of extracellular part of nAChR (b). (a) Polymorphism of RGP residues: 2649 mature RGP sequences downloaded from Genbank were submitted to the VIPR website and the polymorphism was determined and plotted over the length of the mature RGP. Residue 183 is the most polymorphic of all residues within the ectodomain (residues 1–440 in the mature RGP) of the protein. The inset represents the neurotoxin-like peptide domain used in this study. (b) Comparison of K_D values for rabies derived peptides binding to L-AChBP determined by Surface Plasmon Resonance. L-AChBP was immobilized on Biacore CM5 sensor chips and perfused with test peptides at a flow rate of 45 ul/min at 25 degrees centigrade. Average K_D values for at least three independent experiments and standard deviations are shown. Bars indicated significant differences between K_D values of two peptides as determined by Student’s T-test.

Figure 2

Neurotoxin-like domain of rabies virus inhibits nicotinic receptors. Functional effects of rabies glycoprotein neurotoxin-like peptides on α4β2 nAChRs (a–c). Peptides were identical in sequence with the exception of the amino acid present at position 183 (a,b) or 196 (c). Plots shown in (a,b) show inhibition of acetylcholine-induced responses following pre-exposure to peptides containing either a alanine (a) or proline (b) at position 183. Plot (c) shows the effect of a peptide containing an aspartate at position 196 (RV-R196D). The effects of full length ectodomain is shown in panels (d,e). Panel (d) shows the response of oocytes expressing α4β2 nAChRs to co-applications of 30 µM acetylcholine 30 seconds after pre-application of buffer (orange) or RGP ectodomain (blue) at 840 nM concentration. Panel (e) shows the recovery of responses to 30 µM acetylcholine 4 minutes after exposure to RGP ectodomain.

Figure 3

Rabies neurotoxin-like peptide effects on behaviour. (a) Rabies neurotoxin-like peptide inhibits the frequency of nAChR-meditated pharyngeal pumping in C. elegans. C. elegans were injected with peptide and pharyngeal pumping was measured, p < 0.001 by student-T test. (b) Rabies neurotoxin-like peptides alter the behaviour of mice. Mice injected with the peptide were observed and the number of cage transects were determined relative to control injected mice, the horizontal line at 1 represents control injected animals. *Indicate significance at p < 0.1 (*), p < 0.05 (**) or p < 0.01 (***) using two-way ANOVA test. A representative video of injected mice can be seen in supplemental movie.