Binding of neurotoxin-like peptide to homologues of the extracellular domain of nAChR

We used surface plasmon resonance (SPR) techniques to identify the interactions between neurotoxin-like peptides of the RGP and an acetylcholine binding protein derived from Lymnaea stagnalis (L-AChBP). The L-AChBP is a soluble pentameric homolog of the extracellular binding domain of nAChRs and has been used extensively in the study and modelling of these receptors11,12,13,14. Six peptides were used in the SPR assays differing only in the amino acid present at a position corresponding to residue 183 of the mature rabies glycoprotein. This residue has previously been identified as being positively selected in natural rabies isolates15,16 although this positive selection was not found in other studies17. In addition, we determined the polymorphism of residues in over 2600 glycoprotein sequences using the Virus Pathogen Resource (VIPR). Residue 183 is the most polymorphic site in the ectodomain of the rabies glycoprotein while other residues of the neurotoxin-like region are highly conserved among natural rabies viruses (Fig. 1a). A phenotype for mutations at this residue is not known. Rabies virus-derived peptides bound to L-AChBP with dissociation constants (K D ) in the micromolar (μM) range, similar to those observed in previous radioligand binding studies using muscle nAChRs6. We detected significant differences in binding to the L-AChBP with K D values ranging from 3.5 to 73 μM for peptides that differed only at the amino acid corresponding to residue 183 (Fig. 1b). This suggests that this residue is involved in interactions with nicotinic receptors.

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. Full size image

Functional effect of neurotoxin-like peptide on α4β2 nAChR

Next, we performed functional assays on the predominant nAChR subtype in the CNS (α4β2) to determine whether rabies neurotoxin-like region altered the activity of this critical receptor. Assays used two-electrode voltage clamp on nAChRs expressed in Xenopus oocytes. To limit use of vertebrate animals and to focus on the most relevant peptides, we selected the peptides with the two most common amino acids at residue 183 (alanine, and proline), which also had significantly different binding characteristics to ACHBP. A peptide with an alanine at position 183 (RV-183A) inhibited acetylcholine evoked responses on α4β2 nAChR with an extrapolated IC 50 = 465–2620 μM at the 95% CI (Fig. 2a). A peptide with proline at this position (RV-183P) showed slightly higher potency on α4β2 receptors with an extrapolated IC 50 of 185–314 µM (95% CI) (Fig. 2b). The differences between the relative binding to ACHBP and inhibition of α4β2 nAChR between the two peptides are likely due to differences in structure between L-AChBP and the α4β2 subtype of nAChR. In contrast to these two peptides, another variant (RV-R196D, with aspartic acid at position 196) produced no inhibition but instead potentiated responses to acetylcholine (extrapolated EC 50 = 149–605 μM at the 95% CI). However, that peptide did not activate receptors on its own and so is not likely acting as an agonist. Amino acid 196 is an arginine or lysine in naturally occurring RGPs, which corresponds to the quaternary amino group in the acetylcholine molecule and therefore RV-R196D is expected to have reduced activity on the nAChR6. While IC 50 concentrations in the mid micromolar range are higher than those typical of inhibitory ligands at nicotinic receptors in vitro, the limited volume of the synaptic cleft18 results in similar concentrations in vivo based on the estimated 1200 glycoprotein molecules present on a single virus particle19. Indeed, the severity of disease in mice is related to the level of glycoprotein expressed20, indicating that only high glycoprotein concentrations can cause the changes seen. In addition, the relatively small molecular flexibility of the intact glycoprotein as compared to that of the small peptide will likely increase the affinity of the intact protein to the nAChR. To test this, we also assessed the effects of full length RGP-ectodomain on α4β2 receptors. The ectodomain inhibited the receptors at a concentration of 840 nM (Fig. 2d,e). After the application of ACh in the presence of buffer, α4β2 control ACh responses recovered to a level not significantly different (p = 0.168 in a paired t-test, n = 5) from the ACh control responses prior to the buffer application. ACh peak currents were 11.5 ± 1.6 µA and 10.9 ± 1.1 µA prior to and after the buffer application, respectively. However, ACh control responses after the application of the rabies protein (Fig. 2e) were significantly reduced (p = 0.029 in a paired t-test, n = 6) compared to ACh controls obtained prior to the protein application. ACh peak currents were 9.4 ± 3.1 µA and 5.7 ± 1.6 µA prior to and after the protein application, respectively. These results indicate that the neurotoxin-like region, in the context of the full-length protein, has activity at even lower concentrations. Glutamate receptors were not inhibited by the ectodomain at 100 nM while this concentration still significantly inhibited α4β2 receptors compared to buffer treated receptors (data not shown).

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. Full size image

Behaviour modifications by neurotoxin-like peptide

Altered neurotransmitter receptor function could change behaviour in infected hosts. We used the neurotoxin-like peptides instead of full-length ectodomain as the ectodomain could lead to other interactions within the animal that could counteract or alter the nAChR mediated effect. To test the ability of rabies neurotoxin-like peptide to alter nAChR mediated behaviour in an established model organism in vivo we injected Caenorhabditis elegans with neurotoxin-like RPG peptides or peptides with a scrambled sequence and measured pharyngeal pumping. Pharyngeal pumping behaviour in C. elegans is triggered by ACh binding to the nAChR EAT-221. Pharyngeal pumping was absent in six out of seven worms injected with rabies neurotoxin-like peptides (RV-183A). The remaining worm pumped intermittently leading to significantly reduced average pumping frequency (Fig. 3a).

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. Full size image

To test if the RGP neurotoxin-like peptides have effects on behaviour in mammals we injected neurotoxin-like peptide into the cerebrospinal fluid of adult mice to reach an approximate final concentration of 250 micromolar. Mice, with rabies virus-derived peptides (RV-183A or RV-183P) injected into the lateral ventricle, showed increased locomotor activity in an novel-environment behaviour test, when compared to mice receiving control peptide (identical amino acid composition but with a scrambled sequence) or saline (Fig. 3 and supplemental material). Hyperactivity is one clinical sign of rabies22. The peptide RV-R196D that did not inhibit α4β2 nAChR signalling also did not alter mouse behaviour in this assay, showing that behavioural influences of these peptides are related to specific interactions with the nAChR.