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. 2008 Jul 8;105(27):9319-24.
doi: 10.1073/pnas.0801968105. Epub 2008 Jun 30.

Regulation of protein translation through mRNA structure influences MHC class I loading and T cell recognition

Judy Tellam  1 , Corey Smith, Michael Rist, Natasha Webb, Leanne Cooper, Tony Vuocolo, Geoff Connolly, David C Tscharke, Michael P Devoy, Rajiv Khanna
Affiliations

Regulation of protein translation through mRNA structure influences MHC class I loading and T cell recognition

Judy Tellam et al. Proc Natl Acad Sci U S A. .

Abstract

Many viruses avoid immune surveillance during latent infection through reduction in the synthesis of virally encoded proteins. Although antigen presentation critically depends on the level of viral protein synthesis, the precise mechanism used to regulate the generation of antigenic peptide precursors remains elusive. Here, we demonstrate that a purine overloaded virally encoded mRNA lacking secondary structure significantly impacts the efficiency of protein translation and prevents endogenous antigen presentation. Reducing this purine bias through the generation of constructs expressing codon-modified sequences, while maintaining the encoded protein sequence, increased the stem-loop structure of the corresponding mRNA and dramatically enhanced self-synthesis of the viral protein. As a consequence, a higher number of HLA-peptide complexes were detected on the surface of cells expressing this viral protein. Furthermore, these cells were more efficiently recognized by virus-specific T cells compared with those expressing the same antigen expressed by a purine-biased mRNA. These findings delineate a mechanism by which viruses regulate self-synthesis of proteins and offer an effective strategy to evade CD8(+) T cell-mediated immune regulation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Purine bias within the GAr sequence of EBNA1 affects mRNA secondary structure. (A) The native EBNA1 GAr domain has an overrepresentation of purine codons compared to a synthetically generated modified EBNA1 GAr domain where purine bias has been reduced to reflect human average codon usage. (B) Predicted secondary structure of a 400-nt native or codon-modified GAr domain using the mRNA structure analysis program, MFOLD. Free energy values (δG) are shown for both sequences. (C) A series of GAr oligonucteotides were synthesized and inserted into the coding sequence of EBNA1-ΔGA. These engineered GAr domains encode varying lengths of native or codon-modified GAr sequence (100–500 nt).
Fig. 2.
Fig. 2.
A codon-modified GAr sequence influences EBNA1 synthesis. (A) IVT assay of pcDNA3 expression constructs encoding EBNA1 (E1) (lane 1), E1ΔGA (lane 2), E1-GAr(100N) (lane 3), E1-GAr(100M) (lane 4), E1-GAr(200N) (lane 5), E1-GAr(200M) (lane 6), E1-GAr(300N) (lane 7), E1-GAr(300M) (lane 8), E1-GAr(400N) (lane 9), E1-GAr(400M) (lane 10), E1-GAr(500N) (lane 11), or E1-GAr(500M) (lane 12). The constructs were transcribed and translated in vitro with T7 RNA polymerase by using a coupled transcription/translation reticulocyte lysate system. 35S-methionine-labeled proteins were visualized by autoradiography. (B and C) Band intensities from the IVT assay were quantified by densitometric analysis using Imagequant software (Molecular Dynamics) and graphed to demonstrate absolute intensities (B) or relative fold increase of EBNA1 encoded by codon-modified GAr domains compared with EBNA1 encoded by native GAr domains (C). (D) Western blot of EBV-negative HEK293 cells transfected with expression constructs encoding E1-GFP (lane 1), E1ΔGA-GFP (lane 2), E1-GAr(100N)-GFP (lane 3), E1-GAr(100M)-GFP (lane 4), E1-GAr(200N)-GFP (lane 5), E1-GAr(200M)-GFP (lane 6), E1-GAr(300N)-GFP (lane 7), E1-GAr(300M)-GFP (lane 8), E1-GAr(400N)-GFP (lane 9), E1-GAr(400M)-GFP (lane 10), E1-GAr(500N)-GFP (lane 11), or E1-GAr(500M)-GFP (lane 12) with a GFP antibody (Upper) or a monoclonal actin antibody (Lower). Molecular weight markers Mr (kDa) are indicated on the left. (E) Band intensities after immunoblotting were quantified as described for B. Representative data from one of four experiments are presented here.
Fig. 3.
Fig. 3.
Purine bias within the GAr domain decreases the translational efficiency of EBNA1. (A) In vitro transcription assays of EBNA1 encoding either 400 or 500 nt of native or codon-modified GAr sequence demonstrated similar levels of mRNA synthesis. (B) Relative decay of mRNAs for EBNA1-GFP encoding 400 nt of either native or codon-modified GAr sequences, after inhibition of transcription with Actinomycin D. (C) Half-life analysis of E1-GAr(400N)-GFP and E1-GAr(400M)-GFP using FACScan analysis. HEK293 cells, transfected with these expression vectors were treated with 50 μg/ml cycloheximide over a 30-h time course. Expression levels are plotted as a relative percentage of the signal at time 0. (D and E) EBNA1-GFP synthesis rates were measured by transfecting HEK293 cells with either E1-GFP, E1ΔGA-GFP, E1-GAr(300N)-GFP, E1-GAr(300M)-GFP, E1-GAr(400N)-GFP, or E1-GAr(400M)-GFP expression constructs. Transfectants were metabolically labeled overnight in growth medium containing 20 μCi/ml of 3[H]methionine followed by a 30-min pulse with 100 μCi of 35[S]methionine. Cells were lysed, immunoprecipitated with either anti-GFP or anti-tubulin, and subjected to SDS/PAGE. Quantitation of synthesis of these proteins was determined by measuring the 35[S]/3[H] ratio for each protein by liquid scintillation counting (D) and graphed as fold increase in translation rate of modified versus native GAr (E). (F) IVT assay of EBNA1 expression constructs in the presence or absence of a 21-mer antisense oligonucleotide to the native GAr sequence at a molar excess of 10:1 to the GAr. 35S-methionine-labeled proteins were visualized by autoradiography and quantified by densitometric analysis using Imagequant software.
Fig. 4.
Fig. 4.
A codon-modified GAr sequence enhances the presentation of CD8+ T cell epitopes. (A) Expression of surface H-2Kb–SIIN complexes was assessed by flow cytometry on 293KbC2 cells transfected with EBNA1-SIIN-GFP constructs encoding native or codon-modified GAr sequences (as shown). Data are the percent of cells positive for H-2Kb–SIIN when compared with controls cells transfected with relevant parental EBN1-GFP plasmids that lack the SIIN epitope. (B) FLR-specific T cells were incubated overnight in the presence of brefeldin A at a responder to stimulator ratio of 5:1 with fixed EBV-negative SVMR6 cells transfected with selected EBNA1-GFP expression vectors. IFN-γ production by CD3+CD8+ cells was determined by intracellular cytokine staining. (C) HPVGEADYFEY-specific T cells were incubated in duplicate in the presence of brefeldin A for 6 h at a responder-to-stimulator ratio of 5:1 with fixed EBV-negative DG75 cells transfected with selected EBNA1-GFP expression vectors. Cells were incubated with a HPV-specific pentamer followed by anti-CD8 and incubated with anti-IFN-γ and analyzed on a FACSCanto. The top right hand corner of each panel represents the percentage of HPV-specific CD8+ lymphocytes producing IFN-γ. Data shown in C and D are for one representative experiment of three separate experiments.
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Comment in

  • Immune surveillance obstructed by viral mRNA.
    Starck SR, Cardinaud S, Shastri N. Starck SR, et al. Proc Natl Acad Sci U S A. 2008 Jul 8;105(27):9135-6. doi: 10.1073/pnas.0804456105. Epub 2008 Jul 1. Proc Natl Acad Sci U S A. 2008. PMID: 18599434 Free PMC article. No abstract available.

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