Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to G-quadruplexes of EBNA1 mRNA

Abstract

The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1, which is essential for viral genome maintenance but highly antigenic. EBV has seemingly evolved a system in which the mRNA sequence encoding the glycine-alanine repeats (GAr) of the EBNA1 protein limits its expression to the minimal level necessary for function while minimizing immune recognition. Here, we identify nucleolin (NCL) as a host factor required for this process via a direct interaction with G-quadruplexes formed in GAr-encoding mRNA sequence. Overexpression of NCL enhances GAr-based inhibition of EBNA1 protein expression, whereas its downregulation relieves the suppression of both expression and antigen presentation. Moreover, the G-quadruplex ligand PhenDC3 prevents NCL binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression and antigen presentation. Hence the NCL-EBNA1 mRNA interaction is a relevant therapeutic target to trigger an immune response against EBV-carrying cancers.

G-quadruplexes sequester free heme in living cells

“…We therefore designed an experimental strategy that would enable us to detect release of heme from G-quadruplexes by using RNA-Seq analysis to query the effect of treatment with a G-quadruplex ligand predicted to displace sequestered heme on expression of genes involved in heme catabolism and iron homeostasis. We chose to use the well-characterized G4 ligand PhenDC3, a bisquinolinium phenanthroline derivative structurally unrelated to heme (Figure 1A) [De Cian A et al 2007; Piazza A et al 2010; Halder R et al 2012; Chung WJ et al 2014; Perriaud L et al 2014; Lista MJ et al 2017]. PhenDC3 is an ideal compound for genome-wide studies of quadruplex function in vivo , because it readily enters the nucleus [Lefebvre J et al 2017]; it discriminates well between quadruplex and duplex DNA; and it exhibits a very high affinity (nanomolar K d ) for quadruplexes, without pronounced selectivity for any specific quadruplex structure.…”

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].…”

G-Quadruplexes: More Than Just a Kink in Microbial Genomes

“…The identification of G4s in microbial genomes has opened up new avenues for therapeutics; additional studies on the specificity of G4-binding ligands and their undesired effects may help us to better understand the therapeutic potential of this novel group of antimicrobial agents. Host protein-microbial G4 interaction or the host G4-microbial protein interactions at the molecular interface of the host and microbe during infection are fascinating and merit further investigation [55,56,[66][67][68][69]. It would be interesting to understand if such interactions defend the host or demonstrate yet another mechanism of microbial pathogenesis.…”

“…Polysome distribution profiling, in vitro translation, and cell culture experiments identified that formation of quadruplexes obstructs the progress of ribosome machinery, resulting in low levels of EBNA1 and a concomitant decrease in presentation to T cells ( Figure 4D). In addition, nucleolin was identified to bind the G4 formed in the GAr domain of EBNA-1 mRNA [66]. This interaction limits the synthesis of EBNA-1 to levels that allow persistence of the virus and evasion of the host immune system.…”