Christine Tuffereau

Low-affinity nerve-growth factor receptor (P75NTR) can serve as a receptor for rabies virus.

A random‐primed cDNA expression library constructed from the mRNA of neuroblastoma cells (NG108) was used to clone a specific rabies virus (RV) receptor. A soluble form of the RV glycoprotein (Gs) was utilized as a ligand to detect positive cells. We identified the murine low‐affinity nerve‐growth factor receptor, p75NTR. BSR cells stably expressing p75NTR were able to bind Gs and G‐expressing lepidopteran cells. The ability of the RV glycoprotein to bind p75NTR was dependent on the presence of a lysine and arginine in positions 330 and 333 respectively of antigenic site III, which is known to control virus penetration into motor and sensory neurons of adult mice. P75NTR‐expressing BSR cells were permissive for a non‐adapted fox RV isolate (street virus) and nerve growth factor (NGF) decreased this infection. In infected cells, p75NTR associates with the RV glycoprotein and could be precipitated with anti‐G monoclonal antibodies. Therefore, p75NTR is a receptor for street RV.

Arginine or lysine in position 333 of ERA and CVS glycoprotein is necessary for rabies virulence in adult mice

Fixed rabies virus strains (ERA and CVS) produce a fatal paralytic disease in mice after intracerebral or intramuscular injection. Some antigenic mutants of both CVS and ERA viruses with a substitution in position 333 of the glycoprotein (arginine is replaced either by a glutamine, a glycine, or an isoleucine) are totally avirulent for adult mice whatever the dose and the route of inoculation. Here we report an exhaustive investigation of the effect of amino acid 333 on viral virulence. New antigenic mutants were isolated from either CVS, CVS derivatives, or SADBern having arginine in position 333 encoded by CGG, AGG, CGU, or AGA respectively. This study shows that when arginine is replaced by either a leucine, an isoleucine, a methionine, a cysteine, or a serine, the antigenic mutant is also totally avirulent. But when arginine is replaced by a lysine it is still pathogenic although the LD50 by the intracerebral route is higher. Furthermore 41 independent virulent revertants were isolated from four avirulent mutants (with a glycine, a glutamine, a methionine, or a serine in position 333 of the glycoprotein). Thirty-nine regained an arginine at position 333 and 2 had a lysine. From this analysis it appears that the presence of a positively charged amino acid (arginine or lysine) in position 333 of the glycoprotein is necessary for viral virulence.

Rabies virus glycoprotein is a trimer.

Abstract The oligomerization state of the rabies virus envelope glycoprotein (G protein) was determined using electron microscopy and sedimentation analysis of detergent solubilized G. Most of the detergents used in this study solubilized G in a 4 S monomeric form. However, when CHAPS was used, G had a sedimentation coefficient of 9 S. This high sedimentation coefficient allowed its further separation from M1 and M2. Using electron microscopy of negatively stained samples, we studied the morphology of G on virus and after detergent extraction. End-on views of G on virus clearly showed triangles consisting of three dots indicating the trimeric nature of native G. End-on views of CHAPS-isolated G showed very similar triangles confirming that, using this detergent, G was solubilized in its native trimeric structure. Electron microscopy also showed that G had a “head” and a “stalk” and provided the basis for a low-resolution model of the glycoprotein structure.

Rapid sequence evolution of street rabies glycoprotein is related to the highly heterogeneous nature of the viral population

The sequence of the glycoprotein gene of a street rabies virus was determined directly using fragments of a rabid dog brain after PCR amplification. Compared with that of the prototype strain CVS, this sequence displayed 10% divergence in overall amino acid composition. However only 6% divergence was noted in the ectodomain suggesting that structural constraints are exerted on this portion of the glycoprotein. A human strain isolated on cell culture from the saliva of a patient with clinical rabies had only five amino acid differences with the canine isolate, an indication of their close relatedness. These differences could have originated during transmission from dog to dog, or from dog to man, or during isolation on cell culture; they are nonetheless indicative of a genetic evolution of street rabies virus. This evolution was further evidenced by the selection of cell-adapted variants which displayed new amino acid substitutions in the glycoprotein. One of them concerned antigenic site III where arginine at position 333 was replaced by glutamine. As expected this substitution conferred resistance to a site IIIa monoclonal antibody (MAb), but surprisingly did not abolish neurovirulence for adult mice. However, a decrease in the neurovirulence of the cell-adapted variant in the presence of a site IIIa specific MAb was noted, suggesting that neurovirulence was due to a subpopulation neutralizable by the MAb. Simultaneous presence of both the parental and variant sequences was indeed evidenced in the brain of a mouse inoculated with the cell-adapted variant; during multiplication in the mouse brain, the frequency of the parental sequence rose from less than 10% to nearly 50%, indicating the selective advantage conferred by arginine 333 in nervous tissue. Altogether these results were suggestive of an intrinsic heterogeneity of street rabies virus. This heterogeneity was further demonstrated by the sequencing of molecular clones of the glycoprotein gene, which revealed that only one-third of the viral genomes present in the brain of a rabid dog had the consensus sequence. Two-thirds of the clones analyzed displayed from one to three amino acid substitutions. Such heterogeneous populations have been referred to as quasispecies, a concept which implies heterogeneous populations kept together in a dynamic equilibrium. This equilibrium could be rapidly displaced, giving the virus the capacity to adapt easily to new environmental conditions.

Vaccination against rabies : construction and characterization of SAG2, a double avirulent derivative of SADBern

A double avirulent mutant was isolated from the SADBern strain of rabies virus by two successive selection steps using neutralizing anti-glycoprotein monoclonal antibodies. Both mutations affect the triplet coding for amino acid 333 of the glycoprotein. The resulting virus, called SAG2, has a glutamate coded by GAA in position 333 instead of an arginine. This new codon differs by two nucleotides from all the arginine triplets. SAG2 is avirulent in adult mice by intracerebral and intramuscular routes and it protects mice against a challenge by the CVS strain. This double mutant is still avirulent after three successive passages in suckling mouse brain or after ten successive cycles of multiplication in cell culture. Because it is protective and genetically stable, SAG2 represents an improvement on SAG1 which is already used for oral vaccination of foxes in Switzerland and France. It could also be a candidate for oral vaccination of dogs against rabies.

The Rabies Virus Glycoprotein Receptor p75NTR Is Not Essential for Rabies Virus Infection

ABSTRACT Rabies virus glycoprotein (RVG) is known to be the only factor that mediates rabies infection. The neurotrophin receptor (p75NTR), through its cysteine-rich domain 1, is a specific receptor for RVG and neutralizes virus infectivity, but its role in virus infection has remained obscure. We used adult mouse dorsal root ganglion (DRG) neurons as a model to study the role of p75NTR in RV infection of primary neurons. We show that RV infects around 20% of DRG neurons, of which more than 80% are p75NTR positive, have large diameters, and are capsaicin insensitive. Surprisingly, RV binding and infection are absent in about half of the p75NTR-expressing DRG neurons which have small diameters and are often capsaicin sensitive. This indicates that p75NTR is not sufficient to mediate RV interaction in sensory neurons. The rate and specificity of neural infection are unchanged in RV-infected p75NTRExonIV−/− mice that lack all extracellular receptor domains and in wild-type mice infected with two independent RV mutants that lack p75NTR binding. Accordingly, the mortality rate is unchanged in the absence of RV-p75NTR interaction. We conclude that although p75NTR is a receptor for soluble RVG in transfected cells of heterologous expression systems, an RVG-p75NTR interaction is not necessary for RV infection of primary neurons. This means that other receptors are required to mediate RV infection in vivo and in vitro.

Interaction of lyssaviruses with the low-affinity nerve-growth factor receptor p75NTR.

The low-affinity nerve-growth factor receptor p75NTR interacts in vitro with the rabies virus (RV) glycoprotein and serves as a receptor for RV. The Lyssavirus genus comprises seven genotypes (GTs) of rabies and rabies-related viruses. The ability of p75NTR to interact with the glycoprotein of representative lyssaviruses from each GT was investigated. This investigation was based on a specific binding assay between BSR cells infected with a lyssavirus and Spodoptera frugiperda (Sf21) cells expressing p75NTR on the cell surface. A specific interaction was observed with the glycoprotein of GT 1 RV (challenge virus standard or Pasteur virus strains) as well as wild-type RV and the glycoprotein of GT 6 European bat lyssavirus type 2. In contrast, no interaction was detected with the glycoprotein of lyssaviruses of GTs 2-5 and 7. Therefore, p75NTR is only a receptor for some lyssavirus glycoproteins, indicating that the other GTs must use an alternative specific receptor.

Avirulent mutants of rabies virus and their use as live vaccine

Oral vaccination of foxes against rabies began in Switzerland some 20 years ago and was later extended to several European countries. The vaccine strains, which were derivatives of the SAD strain of rabies, retain a non-negligible pathogenicity for rodents and nontarget species. Antigenic mutants of the SAD Bern vaccine strain, which are avirulent for adult mice, foxes and dogs, have been isolated and are presently under trial.

Fatty acylation of rabies virus proteins

The fatty acylation of rabies virus (CVS strain) proteins was investigated. [3H]palmitic acid was found to be incorporated into the glycoprotein G and to a lesser extent into the membrane-associated protein M2. The fatty acid linkage on G was sensitive to sodium borohydride, mercaptoethanol, and hydroxylamine, indicating that the linkage was of the thiolester type. Bromelain digestion indicated that the palmitoylation site on G was located in the intracytoplasmic domain or in the transmembrane domain in which there is only one cysteine in position 461. Therefore, palmitoylation is likely to occur at this position. In the case of M2, the linkage was also sensitive to hydroxylamine and sodium borohydride and to a lesser extent to mercaptoethanol, suggesting that the linkage also occurred on a cysteine.

Phosphorylation of the N and M1 Proteins of Rabies Virus

Phosphorylation of rabies virus proteins was followed in vivo and in vitro. The N and M1 proteins were both found to be phosphorylated. The M1 protein was present in the virion in two phosphorylated states, but only the hypophosphorylated form of M1 was found in infected cells. The hypothesis that some of the M1 molecules become hyperphosphorylated during the maturation process by a membrane-bound kinase was examined. The phosphorylation of the viral proteins by the kinase present in purified rabies virions was studied using an in vitro transcriptase assay: under the conditions of the assay, additional phosphate groups were rapidly attached to the N protein. The M1 protein was similarly hyperphosphorylated although more slowly. Whether the hyperphosphorylation of the N protein is responsible for the poor efficiency of the in vitro transcriptase reaction is not clear. No detectable change in the phosphorylation of cellular proteins was observed in the course of rabies virus infection.