Mutations Conferring Resistance to Neutralization by a Soluble Form of the Neurotrophin Receptor (p75NTR) Map outside of the Known Antigenic Sites of the Rabies Virus Glycoprotein

Abstract

The neurotrophin receptor (p75NTR) serves as a receptor for rabies virus (RV). We expressed and purified a soluble chimera consisting of the p75NTR ectodomain fused to the human immunoglobulin G1 (IgG1) Fc fragment (p75-Fc). Although p75-Fc interacts with RV, the infectivity of RV did not decrease significantly when it was incubated in the presence of the soluble receptor alone. However, when it was subsequently incubated with an antihuman IgG directed against the Fc fragment of p75-Fc, the infectivity of RV was significantly lowered (>90%), whereas incubation with antihuman IgG alone had no effect. We then selected eight independent RV mutants that were not neutralized by p75- Rabies virus glycoprotein (RVG) is the only surface-exposed viral coat protein. It is organized in trimers (21) that protrude from the lipid envelope. RVG has three major functions in the RV infection process. First, RVG mediates cell binding through interaction with cellular receptors and is the first determinant of Lyssavirus neurotropism (3). Second, RVG mediates fusion between viral and cell membranes through a pH-dependent process (22,39). Third, RVG, the main viral antigen, induces both humoral (neutralizing antibodies) (66) and cellular immune responses.RVG is a 505-amino-acid type I membrane glycoprotein (1, 70) with three potential N-glycosylation sites: the first one, located at amino acid 37, is very inefficiently glycosylated (50); the second one, at position 204 or 247, is glycosylated in some strains, but not in others; and the third one, located at amino acid 319, is always glycosylated (67). RVG folding, intracellular transport, and oligomerization have been analyzed for Pasteur virus (PV) and Evelyn Rokitnicki-Abelseth (ERA) virus, and it has been shown that RVG acquired its trimeric organization in the endoplasmic reticulum (18, 65). The putative fusion peptide has also been localized between amino acids 102 and 179 (17).RVG has been extensively mapped with specific anti-RVG monoclonal antibodies (MAbs) by different laboratories. Our group has selected about 50 RVG mutants from the challenge virus strain (CVS) that are resistant to neutralization by anti-RVG MAbs. Several independent antigenic sites have been defined, depending on their interaction with neutralizing anti-RVG MAbs and on the effect of mutations (6, 29). Site II is the major antigenic site (70% of the MAbs recognize this region). It contains two stretches of amino acid residues located at positions 34 to 42 and 184 to 210 (43) joined by a disulfide bridge between cysteines 35 and 207 (15,17,62). Site III is a conformational site (amino acids 330 to 340) and plays a major role in RV propagation in the nervous system and pathogenicity (12,16,49,59). Two minor sites (site a at amino acids 342 and 343 [6] and a linear site at amino acids 251 to 264 [14,28,60]) and several other epitopes have also been described (28). RVG can adopt at least two conformations (20): N (for neutral) and I (for fusion inactive). In the I state, RVG is no longer recognized by site I...

The Rabies Virus Glycoprotein Receptor p75 NTR Is Not Essential for Rabies Virus Infection

“…Using an expression cloning strategy, we have previously shown that p75 NTR can serve as a receptor for RVG (50). We have also show that a soluble form of p75 NTR can bind to RV and neutralizes its infectivity (31), indicating that p75 NTR is sufficient for RVG interaction and that no coreceptor is required. In the present study, we found that RV infection is restricted to a subpopulation of large-diameter, capsaicin-insensitive neurons and that the vast majority of infected neurons express p75 NTR .…”

“…First, p75 NTR might require a cofactor that is not found in these neurons. This is unlikely, because we have shown that a soluble form of p75 NTR is able and sufficient to interact with RV and RVG on its own (31,46) and its affinity constant is 2 nM (46). Second, binding of RV is prevented, and there are several molecules which are tightly associated with p75 NTR and could modify its binding affinity (14).…”

“…The same conclusion was reached by investigations using mutant RV in wild-type mice. We used two different CVS mutants that harbor a point mutation (histidine 352 replaced by arginine [H352R] or phenylalanine 318 replaced by valine [F318V]) in the glycoprotein and do not bind to p75 NTR (31). Wild-type animals were inoculated in the forelimb and monitored for disease progression.…”

“…To unequivocally test the role of p75 NTR in mediating infection, we used two independent strategies. First, we used two strains of knockout mice lacking the extracellular domains of p75 NTR , and second, we investigated mutant virus lacking binding to p75 NTR (31) to determine whether RV mediates infections through p75 NTR .…”

“…TRP channels have emerged as main determinants for the functional heterogeneity of DRG neurons, and the capsaicin receptor TRPV1 is the crucial receptor in the pain pathway and conveniently allows in culture the separation of small-diameter nociceptive sensory neurons from large-diameter nonnociceptive neurons (8). Here, we used two lines of mutant mice lacking p75 NTR and two RV mutants that do not bind to p75 NTR (31) to systematically investigate the importance of RVG-p75 NTR interaction in vivo and in vitro for RV binding and infections.…”