Epstein Barr Virus-Encoded EBNA1 Interference with MHC Class I Antigen Presentation Reveals a Close Correlation between mRNA Translation Initiation and Antigen Presentation

Viruses are known to employ different strategies to manipulate the major histocompatibility (MHC) class I antigen presentation pathway to avoid recognition of the infected host cell by the immune system. However, viral control of antigen presentation via the processes that supply and select antigenic peptide precursors is yet relatively unknown. The Epstein-Barr virus (EBV)-encoded EBNA1 is expressed in all EBV-infected cells, but the immune system fails to detect and destroy EBV-carrying host cells. This immune evasion has been attributed to the capacity of a Gly-Ala repeat (GAr) within EBNA1 to inhibit MHC class I restricted antigen presentation. Here we demonstrate that suppression of mRNA translation initiation by the GAr in cis is sufficient and necessary to prevent presentation of antigenic peptides from mRNAs to which it is fused. Furthermore, we demonstrate a direct correlation between the rate of translation initiation and MHC class I antigen presentation from a certain mRNA. These results support the idea that mRNAs, and not the encoded full length proteins, are used for MHC class I restricted immune surveillance. This offers an additional view on the role of virus-mediated control of mRNA translation initiation and of the mechanisms that control MHC class I restricted antigen presentation in general

Etude des mécanismes moléculaires permettant à la protéine EBNA1 du virus Epstein-Barr d'échapper au système immunitaire

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

In cellulo protein-mRNA interaction assay to determine the action of G-quadruplex-binding molecules

Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions

Domain 1 of the c-myc IRES is responsible for the effect of the c-myc IRES on GAr-dependent translation control.

<p>A) Cartoons illustrating the predicted structure and functional domains of the human c-myc IRES and domains 1 and 2 and the ribosome entry window are indicated. B) Domain 1 was deleted while retaining the ribosome entry window and domain 2 (Δc-myc IRES). C) Autoradiograph of ³⁵S-methionine metabolic pulse labelling and presentation of SL8 derived from the indicated constructs in H1299 cells. Insertion of the Δc-myc IRES in the 5′UTR of the GAr-Ova mRNA (Δc-myc-GAr-Ova) does not restore translation as compare to the intact c-myc IRES (c-myc-GAr-Ova). Neither Δc-myc IRES nor c-myc IRES affect translation when inserted in the 5′UTR of Ova alone. Data are representative of three or more independent experiments with SD.</p

GAr suppresses antigen presentation by targeting the mRNA translation initiation process.

<p>A) Cartoon illustrating the c-myc Internal Ribosomal Entry Sites (IRES) constructs. IRESs offer alternative cap-independent mechanisms of mRNA translation initiation. B) Autoradiograph of ³⁵S-methionine metabolic pulse labelling. Insertion of the c-myc IRES in the 5′UTR of the GAr-Ova mRNA (c-myc-GAr-Ova) restores translation in H1299 cells but does not affect translation when inserted in the 5′UTR of Ova alone (left panel). Western blot shows that the effect of the c-myc IRES is restricted to the GAr alone (right panel). C) Autoradiograph of a 30 minutes ³⁵S-metabolic pulse label experiment of the endogenous protein (actin) and the exogenous GFP protein in H1299 cells in the presence of the c-myc IRES and the GAr constructs. D) The c-myc IRES stimulates SL8 presentation in the context of the GAr from the main open reading frame as well as cryptic translated products (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001151#ppat-1001151-g002" target="_blank">Fig. 2A</a>). Data are representative of three or more independent experiments plus SD.</p

Inhibition of EBNA1 synthesis prevents presentation of peptides derived from the EBNA1 mRNA.

<p>A) Cartoon illustrating the different constructs. The location of the exogenous antigenic peptide sequence SIINFEKL (SL8) of the chicken ovalbumin (Ova) in the EBNA1-SL8 and the EBNA1ΔGA-SL8 constructs is indicated. B) The presentation of SL8 peptide on endogenous MHC class I K^b molecules on (0.5×10⁵) EL4 cells (left) or on human cells co-expressing a genomic K^b construct (right) was determined using B3Z CD8⁺ T cells <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001151#ppat.1001151-Karttunen1" target="_blank">[23]</a>. The GAr domain suppresses presentation of SL8 by over 90% in either cell type. C) Autoradiograph of a 1 hour ³⁵S-methionine pulse label in the presence of proteasome inhibitors shows that the EBNA1-SL8 mRNA is translated 60% less efficiently as compared with the EBNA1ΔGA-SL8. The graph below shows values determined from phosphoimager analysis. D) Western blot shows the steady state level of expression of indicated constructs without proteasome inhibitors. E) Dose-response curve shows that approximately 8 µg of GAr-Ova cDNA is required to reach a similar level of SL8 presentation as that of 1 µg of Ova (left panel). Increasing number of EL4 cells expressing indicated constructs in the presence of a fixed amount (5×10⁴) of B3Z (right graph). The results show representative data from at least three independent experiments in which transfected cells were split and tested for protein synthesis or antigen presentation with SD.</p

The rate of mRNA translation initiation directly correlates with the amount of antigen presented from a given mRNA.

<p>A) A 30 amino acid GAr sequence from the EBV-encoded EBNA1was fused to the N-terminus of Ova (30GAr-EBV-Ova). The GAr sequence from the EBNA1-like protein of the Papio virus carries four single inserted serine residues (30GAr-Papio-Ova). B) Autoradiograph of a 30 minutes ³⁵S-methionine pulse label. C) Presentation of SL8 derived from corresponding constructs in EL4 cells. D) The p53 protein is targeted for the ubiquitin-dependent degradation pathway by the E3 ligase MDM2 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001151#ppat.1001151-Honda1" target="_blank">[42]</a>. Fusion of the Papio GAr to p53 results in an accumulation of polyubiquitinated products in the presence of MDM2, showing that the Papio GAr retains the capacity to affect protein degradation <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001151#ppat.1001151-Daskalogianni1" target="_blank">[24]</a>. E) The GAr sequence consists of single alanines separated by one, two or three glycines. Introducing 2 alanines (GCC) next to each other on 3 separate places (32GAr-3A-Ova) does not alter the overall GC content of the RNA sequence. F) Introducing a single serine next to an alanine at two locations (31GAr-2S-Ova) is more disruptive in terms of mRNA translation as compared with the 32GAr-3A-Ova (left, upper panel). The corresponding effect on antigen presentation is shown in the graph below. The right graph shows the arbitrary values of the rate of mRNA translation initiation and antigen presentation. Data are representative of three or more independent experiments and values are shown with SD. Cells were transfected with the indicated constructs before split and tested separately for antigen presentation or synthesis.</p

mRNA Translation Regulation by the Gly-Ala Repeat of Epstein-Barr Virus Nuclear Antigen 1▿

The glycine-alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis. However, the underlying mechanism of this function remains largely unknown. Here, we have further investigated the effect of the GAr as a regulator of mRNA translation. Introduction of silent mutations in each codon of a 30-amino-acid GAr sequence does not significantly affect the translation-inhibitory capacity, whereas minimal alterations in the amino acid composition have strong effects, which underscores the observation that the amino acid sequence and not the mRNA sequence mediates GAr-dependent translation suppression. The capacity of the GAr to repress translation is dose and position dependent and leads to a relative accumulation of preinitiation complexes on the mRNA. Taken together with the surprising observation that fusion of the 5′ untranslated region (UTR) of the c-myc mRNA to the 5′ UTR of GAr-carrying mRNAs specifically inactivates the effect of the GAr, these results indicate that the GAr targets components of the translation initiation process. We propose a model in which the nascent GAr peptide delays the assembly of the initiation complex on its own mRNA

Antigenic peptides (A.P.) can be derived from the main open reading frame as well as cryptic peptides from alternative reading frames (yellow) and from the 3′UTR (pink) [<b>25</b>].

<p>The nascent GAr polypeptide (red) of the EBNA1 prevents translation initiation throughout the entire mRNA, including its own reading frame and cryptic peptides. This allows the EBV to evade the MHC class I restricted antigen presentation of peptides from the EBNA1 message and helps the virus to evade the immune system. The GAr also prevents the synthesis of the EBNA1 full length protein but its long half life ensures that functional levels of EBNA1 are expressed.</p

GAr-dependent inhibition of antigen presentation in different cell lines from different origins.

<p>The presentation of SIINFEKL from chicken ovalbumin (Ova) itself or Ova inserted in the EBNA1 coding sequence was detected using the B3Z reporter cells. The presentation of SIINFEKL from Ova or from an EBNA1 construct that lacks the GAr sequences (EBNA1ΔGA) was given the arbitrary value of 100%. The right column shows the effect of the c-myc IRES on GAr-dependent inhibition of antigen presentation in cell lines from different origins. The table shows data from at least three experiments and SD. The c-myc IRES overrides GAr-dependent antigen presentation in human derived cell lines only. (N.T.  =  not tested).</p