Posttranslational side chain modification of a viral epitope results in diminished recognition by specific T cells

Abstract

A stretch of 16 amino acid residues within the nominal phosphoprotein of rabies virus was shown to carry an immunodominant epitope for class I-and class II-restricted T cells. The nominal phosphoprotein of rabies virus is thought to be heterogeneously phosphorylated at multiple serine and threonine residues. The synthetic peptide that expressed the T-cell epitope contained a single serine residue corresponding to position 196 of the protein. Phosphorylation of this serine within the synthetic peptide caused a significant decrease of the antigenic potency of the peptide. A similar effect was seen if the serine was replaced by an alanine or if the peptide was glycosylated at its acidic residues. These data suggest that T-cell-mediated recognition of antigen presented by major histocompatibility complex class Ior II-positive cells is impaired not only by point mutations but also by posttranslational side chain modifications of residues within viral epitopes. Little is known about the effect of side chain modifications on recognition by T cells. Previous studies suggested that inhibition of glycosylation of viral antigens might influence T-cell recognition (8, 25). In recent years, numerous viral epitopes have been identified for both class I-and class II-restricted T cells (13, 18, 23). Although many of these viral proteins carry modified side chains under physiological conditions, a review of the literature reveals that, in general, the epitopes identified to date lack natural glycosylation or phosphorylation sites, suggesting that these sites might not be readily accessible for recognition by T cells. The effect of side chain modification was studied by using the nominal phosphoprotein (NS protein [this protein was initially thought to be a nonstructural protein, hence the abbreviation NS]) of rabies virus, a 297-amino-acid protein that is thought to be heterogeneously phosphorylated at multiple serine and threonine residues (4, 6), as been demonstrated for vesicular stomatitis virus, the prototype virus of the Rhabdoviridae family, which includes rabies virus. MATERIALS AND METHODS Mice. Female C3H/He mice were purchased from The Jackson Laboratories, Bar Harbor, Maine, or from Harlan Sprague-Dawley, Indianapolis, Ind. They were immunized at between 8 and 12 weeks of age. Cell lines. The murine L929 fibroblast line (H-2k) was maintained in vitro in Dulbecco's minimal essential medium (MEM) supplemented with 50 jig of gentamicin sulfate per ml, 10 U of penicillin G per ml, 10 ,ug of streptomycin sulfate per ml, and 10% heat-inactivated fetal bovine serum (FBS). BHK-21 cells and HeLa cells (ATCC CCL-2; American Type Culture Collection, Rockville, Md.) were maintained in Eagle's MEM supplemented with 5% FBS and 50 ,ug of gentamycin sulfate per ml. HT-2 cells were maintained in Dulbecco's MEM supplemented with 10% FBS, 10-5 M 2-mercaptoethanol (2-ME), and 10% rat concanavalin A (ConA) supernatant. Viruses. The rabies virus strain Evelyn-Rokitniki-Abelseth

Recognition of the minimal epitope of monoclonal antibody Tau‐1 depends upon the presence of a phosphate group but not its location

“…A few amino acid shift in the stretch of the epitopic residues after phosphorylation was found earlier for polyclonal antibodies anti-T3 and anti-T3P (Lang et al, 1992b). Similar shift in the binding to major histocompatibility complex was observed for the major T cell epitopic peptide of the phosphoprotein of rabies virus (Larson et al, 1992). The identification and the determination of the number of the required phosphate groups of the minimal epitope for mAb AT8 is in progress.…”

Phosphorylated Peptides Are Naturally Processed and Presented by Major Histocompatibility Complex Class I Molecules in Vivo

“…These modifications include deamidation of asparagine (10,11), cysteinylation of cysteine (12)(13)(14), and glycosylation (15). Posttranslational modification alters the ability of T lymphocytes to recognize MHC class I-or class IIrestricted epitopes (10,(12)(13)(14)(15)(16)(17). Recently, Hogan et al (18) identified the only published example of a naturally processed and presented phosphorylated peptide associated with any MHC molecule.…”

Studies on the Rabies Virus RNA Polymerase: 2. Possible Relationships between the Two Forms of the Non‐Catalytic Subunit (P Protein)

“…Fine structures of the rabies virus P protein, however, have not yet been studied precisely, except for some reports that described possible binding domains involved in N-P complex formation and their possible locations at both the N-and C-terminal portions of P protein (9,15). Phosphorylated amino acids of the P protein were identified as serine and threonine (22), but the phosphorylation sites have not yet been identified. Functions of the rabies virus P protein are also thought to be regulated by phosphorylation and dephosphorylation, which however, has not yet been evidenced.…”