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

Abstract

Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs.

Figure 1. Identification and characterization of G-quadruplex structure within the EBNA1 GAr mRNA

(a) A guanine tetrad formed by the coplanar arrangement of four guanine bases held together by Hoogsteen hydrogen bonds and stabilized by a cation (usually potassium, depicted in orange). (b) Schematic representation of EBV EBNA1 depicting the domains essential for genome maintenance functions. Highlighted within the internal GAr domain are the positions of g4-EBNA1 G-quadruplex motifs identified in the corresponding mRNA. The g4-EBNA1 schematic depicts an EBNA1 intramolecular parallel G-quadruplex stabilized by the stacking of two guanine tetrads (gray squares represent guanines as depicted in a). (c) Characterization of the g4-EBNA1 G-quadruplex by CD and UV spectroscopy. The CD (unbroken line) and normalized thermal difference (dashed line) spectra of g4-EBNA1 in the presence of K⁺. (d) CD titration spectra of a 300-nucleotide native EBNA1 GAr transcript (GArN) and a 300-nucleotide codon-modified EBNA1 GAr transcript (GArM) with increasing KCl concentration (0–100 mM).

Figure 2. Codon modification enhances EBNA1 mRNA translation by destabilizing G-quadruplex structures

(a) Representative polysome gradient profile absorbance trace at 254 nm shows the position of polysomes, ribosomes, (80S) and subunits (60S, 40S). (b) Polysome distribution profiles comparing the translation of E1-WT and E1-ΔGAr. (c) Polysome distribution profiles comparing the translation of EBNA1 transfectants expressing 500 nucleotides of native GAr mRNA, E1-GArN and the codon-modified GAr mRNA E1-GArM. (d) Polysome profile of the endogenous GAPDH mRNA observed by RT-qPCR for each EBNA1 variant. (e) IVT assay of EBNA1 expression constructs expressing 300 nucleotides of native GAr mRNA, E1-GArN or the codon-modified GAr mRNA E1-GArM (Supplementary Fig. 8). Band intensities were quantified by densitometric analysis (data represent mean values ± s.e.m.; n = 3). (f) Autoradiograph showing the relative mRNA translation efficiencies of GFP-E1-WT, GFP-E1-ΔGAr and GFP-EBNA1 encoding 500 nucleotides of native GAr mRNA, GFP-E1-GArN or the codon-modified GAr mRNA GFP-E1-GArM. HEK293 cells transfected with GFP-EBNA1 vectors were pulsed with [³⁵S]methionine, and lysates were immunoprecipitated with antibodies to GFP and subjected to SDS-PAGE and autoradiography. M_r, molecular weight markers.

Figure 3. The antisense oligonucleotide AS2 destabilizes g4-EBNA1 G-quadruplexes to enhance EBNA1 expression

(a) AS2 unfolded g4-EBNA1 in a concentration-dependent manner. Time course of the fluorescence intensity of dual-labeled g4-EBNA1 (200 nM) with increasing AS2 concentration (0.25-10 molar equiv.). g4-EBNA1 was labeled with 6-FAM and TAMRA at its 5′ and 3′ ends (excitation at 494 nm and emission at 580 nm). (b) Expansion of the ¹H NMR spectra of g4-EBNA1 when titrated with increasing AS2 concentration. Hoogsteen imino peaks disappeared during AS2 titration in favor of Watson-Crick imino peaks, characterized by a chemical shift (Δ) in the range 12.0-13.0 p.p.m., demonstrating conversion of g4-EBNA1 structure to double-stranded structure. dsRNA, double-stranded RNA. (c) CD spectra of a 300-nucleotide native GAr transcript, GArN, in the absence (bold blue line) or presence (thin blue line) of AS2 (2 equiv., 5 equiv. and 5 equiv. after a 12-h equilibration). The CD spectrum of a 300-nucleotide codon-modified GAr transcript, GArM, is shown for comparison (red line). (d) IVT assays of EBNA1 constructs expressing 400 nucleotides of native GAr, E1-GArN, the codon-modified GAr E1-GArM or a G-quadruplex negative EBV-EBNA3A control construct in the presence and absence of dAS2 at increasing molar excess or an antisense deoxyoligonucleotide to EBNA3A (dAS-E3) (Supplementary Fig. 10). M_r, molecular weight markers. (e) Densitometric quantification of EBNA1 expression in EBNA1-expressing HEK293E cells following transfection with dAS2, dSS1 or a random deoxyoligonucleotide and detected by immunoblotting with antibodies to EBNA1 or β-actin. Band intensities determined the percentage of relative EBNA1 expression (data represent mean values ± s.e.m.; n = 3; Supplementary Fig. 11a).

Figure 4. The small molecule PDS stabilizes g4-EBNA1 G-quadruplex structure and inhibits EBNA1 translation

(a) Structure of PDS. (b) PDS stabilizes g4-EBNA1 in a concentration-dependent manner in the presence of AS2. Fluorescence intensity (F-F₀) time course of a dual labeled g4-EBNA1 (200 nM) in the presence of 10 molar equiv. of AS2 with increasing PDS (0 – 2 molar equiv.). The g4-EBNA1 sequence is labeled as described in Figure 3a. (c) IVT assay of an EBNA1 construct expressing 400 nucleotides of native GAr mRNA (E1-GArN) in the presence of increasing PDS concentration (Supplementary Fig. 16a). (d) IVT assay of an EBNA1 construct expressing 400 nucleotides of native GAr mRNA (E1-GArN) in the presence of an increasing molar excess of PDS and/or 10 equiv. of dAS2 (Supplementary Fig. 17a). (e) Densitometric quantification of an autoradiograph demonstrating decreased EBNA1 synthesis with increasing PDS concentration (Supplementary Fig. 17b). HEK293 cells transfected with a GFP-EBNA1 construct expressing 500 nucleotides of native GAr mRNA (GFP-E1-GArN) in the presence of increasing PDS concentration were pulsed with [³⁵S] methionine, and cell lysates were immunoprecipitated with antibodies to GFP, followed by SDS-PAGE. Band intensities determined the rate of EBNA1 synthesis (data represent mean values ± s.e.m.; n = 3). Molecular weight markers M_r (kDa) are indicated on the left.

Figure 5. A schematic summarizing the translational control of EBNA1 mRNA by cis-regulatory elements, G-quadruplexes, leading to immune evasion

The Epstein-Barr virus-encoded nuclear antigen 1, EBNA1, a genome maintenance protein expressed in all EBV-associated malignancies, self-regulates its synthesis levels to a degree high enough to maintain viral infection but low enough to avoid immune recognition by host virus-specific T cells. Our study demonstrated that RNA secondary structural elements, G-quadruplexes, within the EBNA1 GAr mRNA were responsible for the cis-acting inhibitory effect on EBNA1 synthesis, directly affecting the endogenous presentation of CD8⁺ T-cell epitopes. Targeting the EBNA1 mRNA with an antisense oligonucleotide complementary to the g4-EBNA1 motif destabilizes the G-quadruplex structures and stimulates EBNA1 synthesis and enhances antigen presentation. Targeting the EBNA1 mRNA with a small-molecule quadruplex ligand (PDS) stabilizes the G-quadruplex structures to inhibit EBNA1 mRNA translation and decrease antigen presentation.