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.
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-Fc and antihuman IgG (srr [soluble receptor resistant] mutants). Each mutant carried a single mutation in the glycoprotein gene leading to one amino acid substitution in the protein. A total of four different substitutions were found. Two of the mutations were located at position 318 (phenylalanine replaced by a serine or a valine residue), and two were located at position 352 (histidine replaced by a tyrosine or an arginine residue). All of the mutations prevented the interaction with p75NTR as either a soluble or a membrane-anchored form. Two mutants (F318S) and (H352R) resulted in the formation of small plaques on BSR cells, probably due to the slower maturation of the glycoprotein. Immunoprecipitation, immunofluorescence, and neutralization assays showed that the four mutated glycoproteins still interacted with representative anti-RV glycoprotein monoclonal antibodies (MAbs), indicating that p75NTR binds outside of the known RV glycoprotein antigenic sites.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D007074:Immunoglobulin G / Q000235:genetics; D009500:Neutralization Tests; D017510:Protein Folding; D011820:Rabies virus / Q000378:metabolism*; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism*; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D006023:Glycoproteins; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C064951:glycoprotein G, Rabies virus