Anne Flamand

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.

n Abstractn n 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.n n

Spread of the CVS strain of rabies virus and of the avirulent mutant AvO1 along the olfactory pathways of the mouse after intranasal inoculation

n Abstractn n After intranasal instillation in the mouse, rabies virus (CVS strain) selectively infected olfactory receptor cells. In the main olfactory bulb (MOB), infection was observed in periglomerular, tufted, and mitral cells and in interneurons located in the internal plexiform layer. Beyond the MOB, CVS spread into the brain along the olfactory pathways. This infection is specific to chains of functionally related neurons but at the death of the animal some nuclei remain uninfected. CVS also penetrated the trigeminal system. The avirulent mutant AvOl, carrying a mutation in position 333 of the glycoprotein, infected the olfactory epithelium and the trigeminal nerve as efficiently as CVS. During the second cycle of infection, the mutant was able to infect efficiently periglomerular cells in the MOB and neurons of the horizontal limb of the diagonal band, which indicates that maturation of infective particles is not affected in primarily infected neuronal cells. On the other hand, other neuronal cells permissive for CVS, such as mitral cells or the anterior olfactory nucleus, are completely free of infection with the mutant, indicating that restriction is related to the ability of AvO1 to penetrate several categories of neurons. From these observations, we concluded that CVS should be able to bind several different receptors to penetrate neurons, while the mutant would be unable to recognize some of them.n n

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.

The CVS strain of rabies virus as transneuronal tracer in the olfactory system of mice.

The sequential distribution of transneuronally infected neurons was studied in the olfactory pathway of mice after unilateral inoculation of the challenge virus standard (CVS) strain in the nasal cavity. A first cycle of viral multiplication was observed in a subpopulation of receptor cells scattered in the main olfactory epithelium and in the septal organ. No viral spread from cell body to cell body was reported even in later stages of infection. The second round of viral replication which took place in the ipsilateral main olfactory bulb at 2 and 2.5 days post-inoculation (p.i.), involved second order neurons and periglomerular cells, known to be directly connected with the axon terminals of receptor cells. Also reported as a result of a second cycle of viral replication, was surprisingly the spread of CVS at 2 and 2.5 days p.i. in bulbar interneurons located in the internal plexiform layer and in the superficial granule cell layer, as well as that of 2 ipsilateral cerebral nuclei, the anterior olfactory nucleus and the horizontal limb of the diagonal band. From day 3, a rapid spread of CVS was suggested by detection of virus in all ipsilateral direct terminal regions of the second order neurons and in most tertiary olfactory projections. The locus coeruleus, a noradrenergic nucleus which sends direct afferents to the olfactory bulb, never appeared immunoreactive. In spite of a certain inability of CVS to infect some neuron types, the virus appears relevant to provide new information regarding the complex network of olfactory-related neurons into the CNS.

Etude Genetique du Virus de la Stomatite Vesiculaire: Classement de Mutants Thermosensibles Spontanes en Groupes de Complementation

SommairenLisolement de mutants spontanes thermosensibles a permis dentreprendre letude genetique du Virus de la Stomatite Vesiculaire. Ces mutants donnent des plages a 30° sur un tapis de fibroblastes de poulet mais nen donnent pas a 39.8 ± 0.2°. Cet article rapporte le resultat de tests de complementation conduisant a classer 71 mutants en 5 groupes.nSummarynSeventy-one spontaneous thermosensitive mutants of Vesicular Stomatitis Virus were isolated and tested for complementation. The spontaneous mutation rate seems to be very high since 2.3% of mutants were found in wild-type clones. Experiments were performed at 30° (permissive temperature) and 39.8° (non-permissive temperature). The mutants can be classified into five complementation groups. 58 mutants fall into the first group.

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.

Genetic evidence for multiple functions of the matrix protein of vesicular stomatitis virus

TsO82, a spontaneous temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV) isolated in chick embryo fibroblasts (CEFs), complements the five prototype ts mutants of the virus. The data presented here indicate that the defect in tsO82 is localized in the M gene. The mutation changed a methionine to an arginine at position 51 of the M protein. Only true revertants could be isolated, and their frequency was low, perhaps due to the type of substitution required to return to the wild-type phenotype. TsO82 does not exhibit hypertranscription, in contrast to the data reported for all of the other ts mutants affected in the M protein. Moreover, tsO82 is conditionally ts, since it grows normally in BHK-21 cells at all temperatures. It exhibits no c.p.e. at the non-permissive temperature in CEFs. Our data argue for multiple functions of the M protein of VSV, the domain affected by the tsO82 mutation possibly being implicated both in the shut-off of cellular RNA synthesis, and for the recognition of a cellular factor required for efficient viral RNA synthesis.

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.