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 EBNA1DeltaGA, respectively), which display distinct differences in translation efficiency. We demonstrate that RDPs constitute a significant proportion of newly synthesized EBNA1 and EBNA1DeltaGA and that the levels of RDPs produced by each of these proteins directly correlate with the translation efficiency of either EBNA1 or EBNA1DeltaGA. As a consequence, a higher number of major histocompatibility complex-peptide complexes can be detected on the surface of cells expressing EBNA1DeltaGA, 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.

Figure 1.

EBNA1 and EBNA1ΔGA translation efficiency and rapidly degrading polypeptides. (A) Detection of EBNA1 rapidly degrading polypeptides. HeLa cells treated with 50 μM of both MG132 and lactacystin during the final 30 min of a 60-min starvation in Met-free media were radiolabeled for 2 min and chased for 5 min. EBNA1-GFP immunoprecipitates were subjected to SDS-PAGE and autoradiograpy. Mol wt markers in kD are shown on the left. Arrows indicate full-length EBNA1-GFP and EBNA1ΔGA-GFP. (B) Measurement of EBNA1 synthesis. HEK293 cells transfected with either EBNA1-GFP or EBNA1ΔGA-GFP were metabolically labeled for 12–14 h in growth medium containing 20 μCi/ml of [³H]methionine followed by a 30-min pulse with 100 μCi of [³⁵S]methionine. Cells were lysed and immunoprecipitated with either anti-GFP or antitubulin. Quantitation of EBNA1-GFP, EBNA1ΔGA-GFP, or tubulin synthesis was determined by measuring the [³⁵S] to [³H] ratio for each protein by liquid scintillation counting. (C) In vitro translation assay of EBNA1 and EBNA1ΔGA. pcDNA3.1 expression constructs encoding either EBNA1 or EBNA1ΔGA were transcribed and translated in vitro with T7 RNA polymerase using a coupled transcription–translation reticulocyte lysate system supplemented with [³⁵S]methionine. Band intensities were quantified by densitometric analysis of the imaging data and graphed.

Figure 2.

Endogenous processing of CD8⁺ T cell epitopes from EBNA1 and EBNA1ΔGA. (A) Detection of H-2K^b–SIIN complexes on the surface of 293KbC2 cells transfected with constructs expressing EBNA1 or EBNA1ΔGA. Data shown are the percentage of GFP-positive events (right axis) and the relative increase in median fluorescence for GFP-positive events from transfectants with SIIN-expressing constructs over controls without SIIN, with the value for EBNA1ΔGA made relative to that for EBNA1, which was set to 1 (left axis). (B) CTL recognition of an endogenously processed HLA B8-inserted epitope (FLR) in EBNA1 and EBNA1ΔGA. SVMR6 keratinocytes infected with a recombinant adenovirus encoding either EBNA1 (Ad-EBNA1) or EBNA1ΔGA (Ad-EBNA1ΔGA) were used as targets in a standard ⁵¹Cr–release assay to assess CTL activity. An HLA B8-restricted FLR-specific CTL clone, LC13, was used as an effector in this assay. The multiplicity of infection was 80:1 for this assay, whereas the E to T ratio varied from 20:1 to 0.25:1. These data are representative of three separate experiments. (C) Ex vivo stimulation of FLR-specific CD8⁺ T cells by EBNA1 and EBNA1ΔGA. HLA B8-positive LCLs were infected with expression vectors encoding EBNA1-GFP or EBNA1ΔGA-GFP (both modified to include FLR encoding sequences; see Materials and methods). These cells were used as stimulators to activate FLR-specific CD8⁺ T cells in fresh PBMCs. Responder to stimulator ratios of 10:1 and 20:1 were used in this assay. After a 6-h incubation, FLR-specific CD8⁺ T cells were assessed for intracellular IFN-γ expression using a Cytofix/Cytoperm kit (BD Biosciences).

Figure 3.

Endogenous processing of EBNA1 epitopes in B cells infected with recombinant EBV. (A) Immunoblot of EBV latent gene expression of B cells immortalized with a wild-type EBV BAC (LCL wtEBNA1) or an EBV BAC carrying a GAr-deleted EBNA1 (LCL EBNA1ΔGA). Expression without and after 96 h of cycloheximide treatment is shown. EBNA1, EBNA-2, and LMP1 immunoblots were probed with the OTX1, PE2, and S12 monoclonal antibodies. EBNA-3 expression was detected with EBV immune human sera. (B) CTL recognition of an endogenously processed HLA B35-restricted epitope (HPV) in LCL wtEBNA1 and LCL EBNA1ΔGA. LCL's transformed with recombinant EBV encoding either EBNA1 or EBNA1DGA (D18 and D11) were used as targets in a standard ⁵¹Cr–release assay to assess CTL activity. An HLA B35-restricted HPV-specific CTL line was used as the effector in this assay. (C) Ex vivo stimulation of HPV-specific CD8⁺ T cells by LCLs transformed with recombinant EBV encoding either EBNA1 or EBNA1DGA (D18). These LCLs were used as stimulators to activate HPV-specific CD8⁺ T cells. Responder to stimulator ratios of 20:1, 40:1, 80:1, and 160:1 were used in this assay. After a 6-h incubation, HPV-specific CD8⁺ T cells were assessed for intracellular IFN-γ expression using a Cytofix/Cytoperm kit (BD Biosciences).

Figure 4.

Effect of MHC–peptide stripping and cycloheximide treatment on ex vivo intracellular IFN-γ production by specific T cells generated to the EBNA1 epitope (HPVGEADYFEY). PBMCs from an HLA B35-positive donor were incubated with HLA B35⁺ve donor LCLs, D11 (encoding either full-length EBNA1 or EBNA1ΔGA), which had been untreated or treated with citrate phosphate (pH 3) buffer, cycloheximide (50 μM), or treated with both cycloheximide (50 μM) and citrate buffer. Data shown represents the relative HPV pentamer-positive population producing IFN-γ.