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 (p75(NTR)), 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 p75(NTR) in RV infection of primary neurons. We show that RV infects around 20% of DRG neurons, of which more than 80% are p75(NTR) positive, have large diameters, and are capsaicin insensitive. Surprisingly, RV binding and infection are absent in about half of the p75(NTR)-expressing DRG neurons which have small diameters and are often capsaicin sensitive. This indicates that p75(NTR) is not sufficient to mediate RV interaction in sensory neurons. The rate and specificity of neural infection are unchanged in RV-infected p75(NTRExonIV-/-) mice that lack all extracellular receptor domains and in wild-type mice infected with two independent RV mutants that lack p75(NTR) binding. Accordingly, the mortality rate is unchanged in the absence of RV-p75(NTR) interaction. We conclude that although p75(NTR) is a receptor for soluble RVG in transfected cells of heterologous expression systems, an RVG-p75(NTR) 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.

FIG. 1.

DRG neurons from wild-type mice infected by RV. (A to C) Neurons cultured for 16 h and subsequently infected for 20 h; (D to E) DRG neurons infected in vivo 6 days earlier and subsequently cultured for 4 h. Pictures were taken from areas containing many infected neurons to illustrate their appearance rather than to provide a representative picture of the percentage of infected cells. (A) Phase-contrast image; (D) bright-field image. (B and E) Immunoreactivity for RV phosphoprotein; (C and F) overlay of both pictures. Only large cells are infected in vitro or in vivo. Bars, 50 μm.

FIG. 2.

DRG neurons cultured for 16 h and subsequently infected with RV (A and B) for 12 h or exposed to RV (C and D), each for 30 min, before further processing. (A and C) Bright-field images; (B and D) fluorescent images. (A and C) Dark brown cells are neurons showing capsaicin-induced cobalt uptake. (B and D) Immunoreactivity for p75^NTR (red, extending through the whole soma) and RV phosphoprotein (blue). Bars, 50 μm.

FIG. 3.

Distribution of subpopulations of DRG neurons cultured for 16 h and subsequently infected with RV (25,000 PFU/μl) for 12 h (A) or incubated with RV (5 × 10⁶ PFU/μl) (B) for 30 min. Note the very similar distribution of neuronal subpopulations. There is partial overlap with neurons expressing p75^NTR immunoreactivity (the total population is represented by the two light gray segments), no overlap with neurons showing capsaicin-induced cobalt uptake (in black), and very few p75^NTR-negative neurons (dashed lines) in the RV binding or RV infection segments (dark gray).

FIG. 4.

Immunostaining of DRG cells for p75^NTR (red) shows surface expression in both large- and small-diameter sensory neurons (asterisk). Large- but not small-diameter p75^NTR-positive cells become infected, as shown by immunoreactivity for RV phosphoprotein (green, arrows). Bar, 10 μm.

FIG. 5.

Cultured DRG neurons. (A) Bright field; (B) vital staining for p75^NTR. Both large (cells 1 and 2) and small (cells 3 and 4) cells are labeled. (C and D) Pseudo-color coding of ratiometric Fura-2 fluorescence at baseline (C) and after 10 s application of 1 μM capsaicin (D). Capsaicin-sensitive cells show a dramatic increase in intracellular calcium (red; cells 3, 4, and 5), whereas there is no change in fluorescence intensity in capsaicin-insensitive neurons. Bar, 10 μm.

FIG. 6.

Cell size histogram of subpopulations of DRG neurons cultured for 16 h and subsequently infected for 12 h, showing the relationship of p75^NTR immunoreactivity (A and C to E), RV infection (B and C), and capsaicin-induced cobalt uptake (E). (A) p75^NTR-expressing neurons are found among small and large neurons. (B and C) Histograms of infected neurons (B) and infected neurons expressing p75^NTR (C) show that only large p75^NTR-positive neurons are infected. (D) Noninfected sensory neurons expressing p75^NTR are small. (E) Many of these noninfected p75^NTR-positive cells show capsaicin-induced cobalt uptake.

FIG. 7.

Percentage of neurons cultured for 16 h and subsequently infected for 20 h with the CVS RV strain in wild-type (WT) mice or in mice lacking p75^{NTRExonIV−/−} or with H352R or F318V CVS mutants in WT mice. There is no significant difference in the infection rate.

FIG. 8.

Identification of infected DRG neurons in tissue sections from cervical spinal levels 30 h after inoculation of the forelimb in vivo. Infection was detected by immunohistochemistry using antinucleocapsid antibodies in wild-type mice (A) or in mice lacking p75^NTRExonIV (B) using the CVS RV strain or in wild-type mice using the H352R CVS mutant (C). Bars, 50 μm.