Influence of translation efficiency of homologous viral proteins on the endogenous presentation of CD8+ T cell epitopes

Abstract

A significant proportion of endogenously processed CD8+ T cell epitopes are derived from newly synthesized proteins and rapidly degrading polypeptides (RDPs). It has been hypothesized that the generation of rapidly degrading polypeptides and CD8+ T cell epitopes from these RDP precursors may be influenced by the efficiency of protein translation. Here we address this hypothesis by using the Epstein-Barr virus–encoded nuclear antigen 1 protein (EBNA1), with or without its internal glycine-alanine repeat sequence (EBNA1 and EBNA1ΔGA, respectively), which display distinct differences in translation efficiency. We demonstrate that RDPs constitute a significant proportion of newly synthesized EBNA1 and EBNA1ΔGA and that the levels of RDPs produced by each of these proteins directly correlate with the translation efficiency of either EBNA1 or EBNA1ΔGA. As a consequence, a higher number of major histocompatibility complex–peptide complexes can be detected on the surface of cells expressing EBNA1ΔGA, and these cells are more efficiently recognized by virus-specific cytotoxic T lymphocytes compared to the full-length EBNA1. More importantly, we also demonstrate that the endogenous processing of these CD8+ T cell epitopes is predominantly determined by the rate at which the RDPs are generated rather than the intracellular turnover of these proteins.

G-quadruplexes regulate Epstein-Barr virus–encoded nuclear antigen 1 mRNA translation

“…These observations are important for antigen presentation as studies from our group and others show that the translational efficiency of viral proteins directly affects the generation of rapidly degrading polypeptides (RDPs), which are the primary source of CD8 + T-cell epitopes 7,9,14,15,18,[37][38][39][40][41][42][43][44] . EBNA1 is an ideal model to demonstrate that CD8 + T-cell epitopes are less efficiently processed from a poorly synthesized wild-type EBNA1 generating fewer RDPs compared with a more efficiently synthesized codon-modified EBNA1 where Gquadruplex structures have been destabilized, with a resultant increase in RDPs.…”

“…Lysates were subjected to SDS-PAGE and autoradiography, as described in ref. 15. IVT assays using E1-GArN were also performed in the presence or absence of a range of antisense, sense strand and control deoxyoligonucleotides (Supplementary Table 2) at molar ratios of 10:1 and 20:1 to the EBNA1 mRNA repeat.…”

“…We therefore examined the impact of dAS2 on the endogenous presentation of a MHC class Irestricted epitope fused to EBNA1. An EBNA1-GFP vector expressing native GAr mRNA and carrying a sequence encoding an H-2K b -restricted ovalbumin epitope, SIINFEKL (E1-GArN-SIIN-GFP) 15 , was transfected into HEK293KbC2 cells together with either dAS2 or a control dSS1, and transfected cells were incubated with a SIINFEKL-specific T-cell hybridoma (B3Z cells) 32 . Transfection together with dAS2 enhanced the presentation of the SIINFEKL epitope and resulted in enhanced activation of B3Z T cells at varying effector/ target cell ratios compared to dSS1 ( Supplementary Fig.…”

“…For the HEK293E cells, the DMEM growth medium was also supplemented with G418 (150 μg/mL). The B cell line (DG75) and a naturally infected lymphoblastoid cell line carrying an EBNA1 sequence from a B95-8 EBV BAC were maintained in RPMI 1640 supplemented with 2 mM l-glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin plus 10% FCS 15 . The LCL/EBV BAC culture medium was supplemented with puromycin (Sigma-Aldrich, St. Louis, Mo., USA) (1 μg/ml).…”

“…5), correlates with an inhibition of mRNA translation and restricted endogenous presentation of CD8 + T-cell epitopes 6 . This purine bias is specifically observed within the region encoding the EBNA1 internal glycine-alanine repeat domain (GAr), which is critical for regulating EBNA1 self-synthesis and thereby for minimizing immune recognition [7][8][9][10][11][12][13][14][15][16][17][18][19][20] . Reduction of purine codons within the EBNA1 GAr domain through codon modification markedly reverses this inhibitory effect 6 .…”