mRNA Structural Constraints on EBNA1 Synthesis Impact on In Vivo Antigen Presentation and Early Priming of CD8+ T Cells

Abstract

Recent studies have shown that virally encoded mRNA sequences of genome maintenance proteins from herpesviruses contain clusters of unusual structural elements, G-quadruplexes, which modulate viral protein synthesis. Destabilization of these G-quadruplexes can override the inhibitory effect on self-synthesis of these proteins. Here we show that the purine-rich repetitive mRNA sequence of Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) comprising G-quadruplex structures, limits both the presentation of MHC class I-restricted CD8+ T cell epitopes by CD11c+ dendritic cells in draining lymph nodes and early priming of antigen-specific CD8+ T-cells. Destabilization of the G-quadruplex structures through codon-modification significantly enhanced in vivo antigen presentation and activation of virus-specific T cells. Ex vivo imaging of draining lymph nodes by confocal microscopy revealed enhanced antigen-specific T-cell trafficking and APC-CD8+ T-cell interactions in mice primed with viral vectors encoding a codon-modified EBNA1 protein. More importantly, these antigen-specific T cells displayed enhanced expression of the T-box transcription factor and superior polyfunctionality consistent with the qualitative impact of translation efficiency. These results provide an important insight into how viruses exploit mRNA structure to down regulate synthesis of their viral maintenance proteins and delay priming of antigen-specific T cells, thereby establishing a successful latent infection in vivo. Furthermore, targeting EBNA1 mRNA rather than protein by small molecules or antisense oligonucleotides will enhance EBNA1 synthesis and the early priming of effector T cells, to establish a more rapid immune response and prevent persistent infection.

G-Quadruplexes in Pathogens: A Common Route to Virulence Control?

“…Furthermore, destabilisation of these G4 structures, following transfection of a vector expressing native GAr mRNA fused to a sequence encoding an ovalbumin epitope, resulted in enhanced antigen presentation of that epitope by a T-cell hybridoma [ 57 ]. These results have been mimicked with the EBNA1 antigen itself in an in vivo mouse model, where EBNA1 mRNA G4 destabilisation resulted in enhanced antigen presentation by dendritic cells and the early priming of CD8+ T cells [ 58 ], demonstrating that reducing the translation efficiency of viral proteins is a key part of the latency program of this gammaherpesviruses.…”

G-quadruplex forming sequences in the genome of all known human viruses: A comprehensive guide

“…The presence of G4s in viruses and their involvement in virus key steps is increasingly evident in most of the Baltimore groups [10,11]. In the dsDNA group, G4s were described in both Herpesviridae and Papillomaviridae families [12][13][14][15][16][17][18][19][20]. In ssDNA viruses, the presence of G4s was reported in the adeno-associated virus genome [21].…”

Macavirus latency-associated protein evades immune detection through regulation of protein synthesis in cis depending upon its glycin/glutamate-rich domain

“…Such observations resulted from a number of studies on EBNA1 over the last decade that showed that in spite of the inhibitory effect of the internal GAr domain, CTL responses directed towards EBNA1 could be readily detected in EBV seropositive individuals [ 7 , 46 , 47 ], and we could observe SIINFEKL-specific CTLs after DNA immunization with aLANA-SIIN ( Fig 10 ). This paradox was resolved following extensive in vitro molecular analyses of the endogenous processing of EBNA1 indicating that CTL epitopes from this protein were predominantly generated from newly synthesized DRiPs rather than from the long-lived pool of stable EBNA1 in EBV-infected B cells [ 23 , 26 – 28 , 41 , 47 ]. These observations have subsequently been further extended to demonstrate that the generation of DRiPs is intrinsically linked to the rate at which proteins are synthesized [ 23 ].…”