Title: Low-affinity nerve-growth factor receptor (P75NTR) can serve as a receptor for rabies virus
Abstract: Article15 December 1998free access Low-affinity nerve-growth factor receptor (P75NTR) can serve as a receptor for rabies virus Christine Tuffereau Corresponding Author Christine Tuffereau Laboratoire de Génétique des Virus, CNRS, 91198 Gif sur Yvette, Cedex, France Search for more papers by this author Jacqueline Bénéjean Jacqueline Bénéjean Laboratoire de Génétique des Virus, CNRS, 91198 Gif sur Yvette, Cedex, France Search for more papers by this author Danielle Blondel Danielle Blondel Laboratoire de Génétique des Virus, CNRS, 91198 Gif sur Yvette, Cedex, France Search for more papers by this author Brigitte Kieffer Brigitte Kieffer Laboratoire Protéines et Récepteurs Membranaires, UPR 9050 CNRS, ESBS, Parc d'innovation, Bld Sébastien Brand, 67400 Illkirch, France Search for more papers by this author Anne Flamand Anne Flamand Laboratoire de Génétique des Virus, CNRS, 91198 Gif sur Yvette, Cedex, France Search for more papers by this author Christine Tuffereau Corresponding Author Christine Tuffereau Laboratoire de Génétique des Virus, CNRS, 91198 Gif sur Yvette, Cedex, France Search for more papers by this author Jacqueline Bénéjean Jacqueline Bénéjean Laboratoire de Génétique des Virus, CNRS, 91198 Gif sur Yvette, Cedex, France Search for more papers by this author Danielle Blondel Danielle Blondel Laboratoire de Génétique des Virus, CNRS, 91198 Gif sur Yvette, Cedex, France Search for more papers by this author Brigitte Kieffer Brigitte Kieffer Laboratoire Protéines et Récepteurs Membranaires, UPR 9050 CNRS, ESBS, Parc d'innovation, Bld Sébastien Brand, 67400 Illkirch, France Search for more papers by this author Anne Flamand Anne Flamand Laboratoire de Génétique des Virus, CNRS, 91198 Gif sur Yvette, Cedex, France Search for more papers by this author Author Information Christine Tuffereau 1, Jacqueline Bénéjean1, Danielle Blondel1, Brigitte Kieffer2 and Anne Flamand1 1Laboratoire de Génétique des Virus, CNRS, 91198 Gif sur Yvette, Cedex, France 2Laboratoire Protéines et Récepteurs Membranaires, UPR 9050 CNRS, ESBS, Parc d'innovation, Bld Sébastien Brand, 67400 Illkirch, France *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:7250-7259https://doi.org/10.1093/emboj/17.24.7250 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info 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. Introduction Rabies virus (RV) is a lyssavirus that belongs to the rhabdovirus family. It is a neurotropic virus usually transmitted through the bite of a rabid animal (Charlton, 1994; Dietzschold et al., 1996). RV penetrates either directly into nerve endings at the site of inoculation (Shankar et al., 1991) or after a limited multiplication in myotubes (Murphy et al., 1973a; Harrison and Murphy, 1978); it is then transported along axons (Tsiang, 1978) to the cell body of motor and sensory neurons, where replication takes place. Viral budding is observed mostly in internal compartments of infected neurons (Gosztonyi, 1994) and the virus is transported to synapses in vesicles. Within the nervous system (NS), propagation of RV between connected neurons occurs exclusively at the synapse. Late in infection, the virus eventually spreads to a few categories of non-neuronal differentiated tissues, such as submaxillary salivary glands, taste buds, adrenal glands, pancreas, kidney, hair follicles and brown fat tissue (Murphy et al., 1973b). At this stage, classic rabies symptoms develop and death occurs rapidly. Apart from the very beginning and end of the infectious process, RV multiplies and propagates exclusively inside neurons. This neuronal tropism in vivo is also observed in vitro with street RV isolates extracted from salivary glands or from the brains of rabid animals. In vitro, such isolates can only infect established cell lines of neuronal origin. However, viruses can be adapted (Kissling, 1958) and several passages are required for the virus to be adapted fully to the in vitro multiplication. Additional cycles of multiplication in non-neuronal cells are necessary for the selection of fixed strains that would multiply in established cell lines such as BHK21, BSR and Vero cells (Wiktor et al., 1964; Schneider et al., 1971). Evelyn Rokitnicki Abelseth (ERA), Pasteur Virus (PV) or Challenge Virus Standard (CVS) are fixed RV strains that have been selected in the past according to this procedure and are used around the world for laboratory investigation. All have kept their specific tropism for neurons in animals and propagate in the nervous system like street viruses. Therefore, adaptation did not abolish neurotropism but rendered the virus able to grow in non-neuronal cells. It is postulated, but not demonstrated, that adaptation is at least partly due to the capability of fixed strains of RV to use ubiquitous receptors present on every cell type investigated to date (Seganti et al., 1990). Ubiquitous receptors could be molecules such as phospholipids (Superti et al., 1984), gangliosides (Conti et al., 1986; Superti et al., 1986) or proteins (Wunner et al., 1984; Broughan and Wunner, 1995; Gastka et al., 1996). Recently, the neural cell-adhesion molecule has been shown to be a receptor for RV laboratory strains (Thoulouze et al., 1998). Also, it has been proposed that the nicotinic acetylcholine receptor (nAChR) serves as a receptor for RV (Lentz et al., 1984, 1986; Hanham et al., 1993). Nevertheless, the fact that RV infects neurons that do not express nAChR (McGehee and Lorca, 1995) suggests the existence of other molecules mediating viral entry into neurons. It should be emphasized that the nAChR is located mainly on muscle cells and could account for the ability of street RV to multiply locally in myotubes at the site of inoculation (Burrage et al., 1985) which would facilitate subsequent penetration into neurons. The RNA genome of RV is 11 930 nucleotides long and of negative polarity. It encodes five proteins. The unique glycoprotein (G) is organized in trimers which protrude from the viral envelope (Whitt et al., 1991; Gaudin et al., 1992). It is a type I integral transmembrane glycoprotein with an external domain of 439 amino acids (aa), a transmembrane region of 22 aa and a C-terminal cytoplasmic domain of 44 aa. Extensive studies of the antigenicity of the protein have identified two immunodominant conformational sites, named sites II and III (Seif et al., 1985; Préhaud et al., 1988), one minor site (site a) (Benmansour et al., 1991) and several linear epitopes (Bunschoten et al., 1989; Raux et al., 1995; Lafay et al., 1996) on the external domain. The G protein is a major determinant of the viral neurotropism. Mutations in the glycoprotein reduce or abolish neuroinvasiveness without impairing the ability of the virus to multiply in cell culture. Replacement of Arg333, situated in site III of the G protein, results in the loss of virulence for adult animals (Dietzschold et al., 1983b; Seif et al., 1985; Tuffereau et al., 1989). The mutant virus is still able to infect peripheral neurons but is only transmitted to a few categories of second order neurons in the central nervous system (CNS) (Dietzschold et al., 1985; Coulon et al., 1989; Lafay et al., 1991). We have shown recently that the additional mutation of Lys330, also in site III, abolishes the penetration of the virus into motor and sensory neurons after intramuscular inoculation of the virus (Coulon et al., 1998). Even if experimental evidence suggests the existence of as yet unidentified neuronal receptors for RV, the presence of ubiquitous receptors sufficient to mediate the penetration of RV into most, if not all, cell lines in vitro is a considerable limitation to the cloning of such receptors. We have recently demonstrated the existence of specific binding sites for the RV glycoprotein in neuronal cell lines of various origins. These sites mediate the fixation of Spodoptera frugiperda (Sf21) cells expressing the RV glycoprotein (G-Sf21 cells) on their surface (Tuffereau et al., 1998). They are not present on the non-neuronal cell lines tested so far. Mutations at positions 330 and 333 of the glycoprotein greatly reduce the binding. These sites are different from nAChR because antibodies directed at this receptor do not abolish binding to G-Sf21 cells. In the present study, we have observed that the truncated form of the glycoprotein (Gs), which is cleaved and secreted naturally from infected cells (Dietzschold et al., 1983a; Morimoto et al., 1993), is also found in the supernatant of G-Sf21 cells. Gs as well as G-Sf21 cells were capable of binding to neuroblastoma cells (i.e. NG108 cells), but not to cell lines of non-neuronal origin such as COS7 or BSR cells. This observation allowed us to use an expression-cloning strategy to identify the protein responsible for Gs and G-Sf21 binding to NG108 cells. We expressed an NG108 library in COS7 cells and obtained a cDNA clone encoding a protein capable of binding Gs. BSR cells stably expressing this protein could be infected by a non-adapted field RV isolate originating from a fox. This virus was unable to grow on BSR cells not expressing the receptor. Sequencing of the isolated clone identified it as the mouse counterpart of the rat and human low-affinity nerve-growth factor receptor (p75NTR; Johnson et al., 1986; Radeke et al., 1987). Results G-Sf21 cells produce a soluble form of the RV glycoprotein In the supernatant of RV-infected BHK cells, a soluble glycoprotein, Gs, has been detected (Dietzschold et al., 1983a; Morimoto et al., 1993). Gs results from the cleavage of the mature G, releasing the ectodomain and the first 8 aa of the transmembrane domain in the supernatant. Gs is antigenically indistinguishable from the G protein in its native configuration (Dietzschold et al., 1983a). We analysed whether Sf21 cells that have been infected with a recombinant baculovirus carrying the RV glycoprotein gene also secreted Gs. Supernatants from four separate batches of infected lepidopteran cells were analysed by SDS–PAGE together with 3, 5 and 10 ng of purified G protein from RV. Proteins were transferred onto nitrocellulose membranes and revealed with an anti-G monoclonal antibody (mAb). In the supernatants of G-Sf21 cells (Figure 1A, lanes a–d), a major protein (Gs) and a minor protein (Gb) were detected. After ultracentrifugation, Gb was associated preferentially with the pellet fraction (Figure 1B, lane P), while Gs remained in the supernatant (Figure 1B, lane S). Therefore, it is probable that Gb is associated with heavier structures, such as membrane debris or vesicles, or that it is inserted in the envelope of recombinant baculoviruses, as already published (Barsoum et al., 1997). Gb produced in lepidopteran cells migrated faster than G produced in mammalian cells, due to the incomplete maturation of the sugar chains in insect cells (Jarvis and Finn, 1995) and due to the presence of bovine serum albumin in the cell medium. Comparison of the intensity of the bands between purified G and Gs suggested that the four batches of G-Sf21 cells (Figure 1A, lanes a–d) secreted ∼0.5 μg/ml of Gs in the supernatant. Gs was recognized by mAbs directed to the known antigenic regions of the native G (data not shown) as described for Gs present in the supernatant from RV-infected BSR cells (Dietzschold et al., 1983a). Figure 1.Analysis of the soluble form of the RV glycoprotein (Gs). (A) Three, 5 and 10 ng of purified G from RV together with 15 μl of four different G-Sf21 cell supernatants were run on a 10% SDS–polyacrylamide gel (lanes a, b, c and d). Western blotting of the gel was performed and G was detected with the anti-G mAb 17D2. (B) After ultracentrifugation of the G-Sf21 culture medium, the pellet fraction (P) was resuspended in a volume equivalent to the initial volume and the supernatant fraction (S) was kept. Fifteen microlitres of each sample were analysed as in (A). Gb: migration of RV G expressed in Sf21 cells. Download figure Download PowerPoint Gs attaches to neuroblastoma cells but not to COS7 cells We have shown previously that G-Sf21 cells attach to various neuroblastoma cell lines through interaction between the RV glycoprotein and neuronal cell-surface molecules (Tuffereau et al., 1998). When neuroblastoma cells, such as NG108 cells, were treated with the supernatant of G-Sf21 cells, they retained Gs at their surface. As a result, cells treated successively with a mixture of mAbs directed against various regions of the RV glycoprotein (see Materials and methods) and then with a β-galactosidase-labelled anti-mouse antibody became blue after X-gal staining. Anti-G mAb 50AD1, which is specific for site III, did not recognize Gs when bound to the NG108 cells but recognized Gs in solution (data not shown). COS7 cells did not retain Gs and were not stained (Figure 2A). When a mixture of NG108 and COS7 cells was incubated with the G-Sf21 supernatant, only NG108 cells, which could be clearly differentiated from COS7 cells by cell morphology, retained Gs (Figure 2B). When NG108 cells were differentiated with Na-butyrate and incubated with the supernatant of G-Sf21 cells, a high density of Gs bound to the nerve processes (Figure 2C). After ultracentrifugation of the G-Sf21 culture medium, supernatant containing Gs gave a heavy staining when incubated with NG108 cells, while the resuspended pellet containing only Gb bound poorly to these cells (not shown). Consequently, the supernatant of G-Sf21 cells was used without further purification. No blue staining of NG108 cells was observed after treatment with culture medium from non-infected Sf21 cells or from Sf21 cells infected with a lacZ-recombinant baculovirus (data not shown). Gs released in the supernatant of RV-infected BSR cells was also able to bind specifically to NG108 cells (data not shown). Figure 2.Binding of Gs to neuroblastoma cells. COS7 cells (A), a COS7 and NG108 co-culture (B) and NG108 cells differentiated for 4 days in the presence of 5 mM Na-butyrate (C) were incubated with 3 ml of the supernatant from G-Sf21 cells collected 2 days after infection and containing ∼1.5 μg of Gs. The cells were washed, fixed with paraformaldehyde, incubated with anti-G mAbs, then with a β-galactosidase-labelled anti-mouse conjugate and finally stained with the X-gal substrate. Magnification: ×150. Download figure Download PowerPoint Identification of a putative receptor for RV using Gs as a ligand Since Gs binds specifically to NG108 cells, it could serve in a screening assay devised at cloning a putative RV receptor. We used a random-primed cDNA library derived from mRNA of NG108 cells. This library contained 2.9×106 primary transformants, up to 85% of the clones had inserts, and half of the inserts were >1500 bp (Kieffer, 1991). This library was used successfully to clone the δ-opioid receptor (Kieffer et al., 1992). In a preliminary experiment, we observed that the transfection of the whole library into COS7 cells resulted in a few cells that expressed a surface molecule able to retain Gs from G-Sf21 cell supernatant (data not shown), indicating that the library contained the gene(s) of interest. It was divided into pools of 1000–1200 different recombinant bacterial colonies. Plasmid DNA from 150 of these pools were used to transfect 2×105 COS7 cells. One pool gave 30–40 cells that were light blue after successive incubation with the G-Sf21 cell supernatant, anti-G mAbs, the anti-mouse antibody and the X-gal substrate. This pool was subdivided twice into subpools of 200 and then eight plasmids. At this stage, 80% of the transfected cells stained blue and the intensity of the staining increased. Ultimate subcloning led to the isolation of a single plasmid (plasmid 8-2). COS7 cells or BSR cells transfected with this plasmid bound Gs and the intensity of the staining was similar (Figure 3A and B). Transfected cells were not stained when the treatment with G-Sf21 cell supernatant was omitted (Figure 3C). Figure 3.Binding of Gs to p75NTR-expressing cells. BSR (A) or COS7 cells (B) transiently transfected with plasmid 8-2 or BSR-R5 cells (D) were stained as described in the legend to Figure 2. (C) Incubation of 8-2-transfected BSR cells with Gs was omitted. Magnification: ×150. Download figure Download PowerPoint Plasmid 8-2 encodes the murine low-affinity nerve-growth factor (NGF) receptor The plasmid 8-2 contained a 1.3 kb insert with a large open reading frame of 1251 bp corresponding to a sequence of 417 aa. The predicted sequence showed high homology with the rat (Radeke et al., 1987) and human (Johnson et al., 1986) low-affinity NGF receptor, p75NTR. It was also homologous, although to a lesser extent, to the chicken receptor (Figure 4). The putative transmembrane protein contains a hydrophobic stretch of 21 aa at its N-terminus, presumably acting as a signal peptide, an external domain of 220 aa containing four cysteine-rich domains and a stalk rich in serine and threonine, a 22 aa transmembrane domain and a 154 aa cytoplasmic domain. The external domain of the protein has one conserved potential N-glycosylation site and the stalk is highly O-glycosylated. There are 14 aa differences between the rat p75NTR and our sequence, which are all located in the ectodomain (five in the cysteine-rich domain and nine in the stalk of the ectodomain). Since the NG108 cell line is a hybrid between a murine neuroblastoma (N18) and a rat glioma (C6) cell line, sequencing of mouse genomic DNA amplified by PCR was performed to ensure the origin of our clone. Comparison between the mouse genomic sequence and the sequence from plasmid 8-2 showed that we have actually cloned the murine p75NTR (data not shown). Figure 4.Alignment of the putative protein sequence encoded by plasmid 8-2 to the rat (r), human (h) and chicken (c) p75NTR. The sequence of the chicken homolog was from Large et al. (1989). The transmembrane domain is underlined. Download figure Download PowerPoint Since the size of the insert in plasmid 8-2 was shorter (1300 bp) than the rat and human p75NTR mRNA (3700 bp), Northern blot analysis was performed. It indicated that the murine p75NTR mRNA in NG108 cells was ∼3700 bp (data not shown), which is in accordance with the mRNA size in rat PC12 cells (Radeke et al., 1987). The insert of the isolated clone, therefore, is missing the long 3′ untranslated sequence of the p75NTR mRNA. COS7 cells transfected with plasmid 8-2 expressed a doublet of proteins of ∼75 kDa which was recognized by Western blotting with a rabbit polyclonal serum directed against the cytoplasmic domain of the human p75NTR (Figure 5A). This serum also recognized a doublet of the same molecular weight in PC12 cell extracts and did not react with COS7 cell extracts transfected with the pCDM8 control plasmid (Figure 5A). In addition, a minor band of low molecular weight was detected. This polypeptide corresponds to the cytoplasmic and transmembrane domains of the protein that have been described in PC12 cells after cleavage of the external domain (DiStephano and Johnson, 1988). Figure 5.Immunodetection of p75NTR. Cell extracts were prepared from PC12 cells or COS7 cells transfected with either plasmid pCDM8 or 8-2 (A) or from the control line C12 and the p75NTR-expressing cells R4 or R5 (B) and analysed by Western blotting using a polyclonal rabbit serum directed against the cytoplasmic domain of the human p75NTR. Truncated forms of p75NTR are indicated with arrow heads. Download figure Download PowerPoint Stable expression of p75NTR in BSR cells To isolate clones of BSR cells stably expressing p75NTR, BSR cells were cotransfected by plasmids 8-2 and pSV2 Neo. After several cycles of multiplication, surviving cells were cloned and ∼30 colonies able to grow in the presence of geneticin were selected. Most of them were able to bind Gs when treated with the supernatant of G-Sf21 cells, although the quantity of Gs attached, and consequently the intensity of staining, varied. The staining of the cells within individual clones was homogenous (Figure 3D). Two of these clones (R4 and R5) were shown to express p75NTR (Figure 5B). Control BSR clones also were isolated after transfection of BSR cells with plasmids pCDM8 and pSV2 Neo. None of these clones were able to bind Gs. One control clone (C12) was selected for further studies. As expected, it did not express p75NTR (Figure 5B). The attachment of G-Sf21 cells on six clones stably expressing p75NTR (R4, R5, R7, R11, R12 and R13) was analysed. The level of fixation varied from clone to clone and remained stable during >30 passages of the cells (Figure 6A). For instance, R7 always showed a low level of G-Sf21 binding, 3–5 times higher than the background, while binding to R5 was high. G-Sf21 cells did not attach to the control BSR clone (C12) and Sf21 cells infected with a LacZ expressing baculovirus were not retained at the surface of receptor-expressing cells (data not shown). We have demonstrated previously that mutations in positions 330 and 333 of the RV glycoprotein abolished the penetration of RV into motor and sensory neurons after intramuscular inoculation of adult mice (Coulon et al., 1998). We have shown also that these mutations greatly decreased the binding of G-Sf21 cells to NG108 cells, although the quantity of mutated G at the surface of the insect cells was not reduced (Tuffereau et al., 1998). These mutations similarly reduced the binding of G-Sf21 to p75NTR-expressing clones (Figure 6B). Figure 6.Binding of G-Sf21 cells to BSR lines expressing p75NTR. (A) The binding assay with Gcvs-Sf21 was performed as described in Materials and methods. Two independent binding experiments were performed at passage 15 (hatched bars) or at passage 40 (open bars). For R13, only passage 40 was analysed. The binding efficiency was expressed as the ratio of the number of bound cells to the total number of insect cells added to the cell monolayers. Each bar represents the average of three determinations with standard deviations (SD). (B) Comparison of binding of G-Sf21 cells expressing either the parental Gcvs glycoprotein (black bars) or the doubly mutated glycoprotein (K330N+R333M) (hatched bars) to BSR cells expressing p75NTR as described in Materials and methods. The binding efficiency was expressed as the ratio of the number of bound cells to the total number of insect cells added to the monolayers. The experiment was done at passage 43. Each bar represents the average of three determinations. Download figure Download PowerPoint Cells expressing p75NTR are permissive for a field RV isolate With the exception of neuroblastoma cells, field RV isolated from the brain of infected animals does not grow in cultured cells. The virus has to be adapted to growth in cultured cells by successive passages in neuroblastoma cells and then in cell lines of non-neuronal origin. We infected p75NTR-expressing cells with a fox isolate of RV that had been multiplied once in suckling mice and once in hamster. The hamster brain extract was titrated at 107 LD50/ml by intracerebral inoculation of adult mice. A 10-fold dilution of this viral suspension was added to R4, R5, R13 and to the control clone C12. After 22 h, cells were fixed and stained with a fluorescein-conjugated anti-nucleocapsid antibody. On p75NTR-expressing cells, numerous positive cells were counted (Table I) and the ratio between infectious units (I.U.) and LD50 in the inoculum was close to 0.1. Considering that the viral suspension was still contaminated heavily with brain material that could have an inhibitory effect on the development of the infectious cycle, it is likely that the ratio could be even higher. As expected, few infected cells were detected on C12 cells and the ratio between I.U. and LD50 in the inoculum was <0.002. The fox RV isolate could also propagate from cell to cell under agarose and gave foci which could be stained with fluorescent antinucleocapsid antibodies after permeabilization of the cell layer (data not shown). Therefore, the presence of p75NTR enabled the penetration of field RV and the development of an efficient infection into otherwise refractory cells. The fixation of field RV was inhibited by the mouse β-NGF, a natural ligand of p75NTR (Figure 7). The inhibition was in the range of 52, 35 and 9% for doses of NGF equal to 100, 33 and 10 nM, respectively. Figure 7.Inhibition of the fixation of a field RV isolate to R5 by NGF. Cells were treated for 30 min at room temperature with 100, 33 or 10 nM of mouse NGF before infection with the G5H field RV as described in the legend to Table II. Cells were incubated at 37°C for 22 h, fixed and stained with anti-nucleocapsid antibodies. Twelve to 15 fields were counted for each NGF concentration. Each bar represents the average of three determinations. Download figure Download PowerPoint Table 1. Infection of BSR cells stably expressing p75NTR with a RV street isolate C12 R4 R5 R13 Experiment 1 25–50 ND 104 6×103 Experiment 2 25–50 2×103 6×103 ND Experiment 3 75–100 5×103 104 ND Experiment 4 100–125 4×103 6×103 ND Infection was performed with 0.1 ml of a 10-fold dilution of an infected brain homogenate titrating 107 LD50/ml onto 9 cm2 slide flasks. Twenty-two hours after infection, the cells were fixed and stained with a fluorescein-labelled anti-nucleocapsid antibody. Fluorescent cells were counted on 10–15 randomly distributed 2 mm2 fields representing ∼2–3% of the flask. Data are expressed as I.U./flask. ND, not done. Interaction of the CVS glycoprotein with p75NTR BSR cells are fully susceptible to the adapted strains of RV and the presence of p75NTR did not significantly increase their susceptibility to infection by these viruses. For instance, the number of CVS plaques counted on the p75NTR-stable lines were only slightly higher than on the control cell line C12 (Table II). When viral infection was performed in the presence of 10% of serum, p75NTR-expressing cells were 3–10 times more susceptible to CVS infection than control cells (data not shown), indicating that p75NTR can serve as a receptor for RV laboratory strains. Viral production of the p75NTR-stable lines after infection by the CVS strain was equivalent or slightly lower than obtained from control cells (data not shown). When C12- or R5-infected cells were labelled, solubilized with detergent and then incubated with anti-p75NTR, some viral G coimmunoprecipitated with p75NTR (Figure 8A, lane R5+), thus indicating a direct interaction between G and its receptor. The inefficient 35S incorporation in p75NTR did not allow its detection by autoradiography when the immunoprecipitation was performed with anti-G mAbs (Figure 8A). However, p75NTR was detected in Western blot analysis of the same immune complexes (Figure 8B). The receptor was not present in immune precipitates from non-infected R5 cell extracts treated with anti-G mAbs. Figure 8.Interaction between p75NTR and the RV glycoprotein. Stable lines C12 and R5 were either not infected (−) or infected (+) at a m.o.i. of 3 for 16 h with the CVS strain of RV. (A) Cells were labelled in the presence of 35S, then solubilized in lysis buffer. Immunoprecipitations were performed with anti-G mAbs (30AA5 + 17D2) or with an anti-p75NTR serum. Immune complexes were analysed by SDS–PAGE (8% acrylamide) followed by autoradiography. A longer exposure of the gel is shown for the immunoprecipitation performed with anti-p75NTR. G from the CVS strain of RV and p75NTR migrate as doublets. (B) Immunoprecipitations of cold extracts with anti-G mAbs were performed as described in (A). Immune complexes together with an extract from R5 cells were analysed by Western blotting with the anti-p75NTR antibody after migration on an 8% SDS–polyacrylamide gel. The asterisk indicates the p75NTR immunoprecipitated by anti-G mAbs. Download figure Download PowerPoint Table 2. Titration of the CVS laboratory RV strain on BSR cells stably expressing p75NTR C12 R4 R5 R13 Experiment 1 1.3×107 ND 3×107 107 Experiment 2 2×107 4.8×107 3.4×107 4.5×107 Experiment 3 2.6×107 4.1×107 2.5×107 2.7×107 Serial dilutions of the CVS strain were performed and used to infect cell monolayers and plaques were counted 4 days after infection. The data are expressed as p.f.u./ml. Discussion RV penetrates into peripheral neurons at the site of inoculation and propagates in the CNS by transynaptic transmission to connected neurons. Direct attempts to identify molecules which could mediate the entry of RV into nerve endings have been impaired by the fact