A yeast-based assay identifies drugs that interfere with Epstein-Barr virus immune evasion

Abstract

Epstein-Barr virus (EBV) is tightly associated with certain human cancers, but there is as yet no specific treatment against EBV-related diseases. The EBV-encoded EBNA1 protein is essential to maintain viral episomes and for viral persistence. As such, EBNA1 is expressed in all EBV-infected cells, and is highly antigenic. All infected individuals, including individuals with cancer, have CD8+ T cells directed towards EBNA1 epitopes, yet the immune system fails to detect and destroy cells harboring the virus. EBV immune evasion depends on the capacity of the Gly-Ala repeat (GAr) domain of EBNA1 to inhibit the translation of its own mRNA in cis, thereby limiting the production of EBNA1-derived antigenic peptides presented by the major histocompatibility complex (MHC) class I pathway. Here we establish a yeast-based assay for monitoring GAr-dependent inhibition of translation. Using this assay we identify doxorubicin (DXR) as a compound that specifically interferes with the GAr effect on translation in yeast. DXR targets the topoisomerase-II–DNA complexes and thereby causes genomic damage. We show, however, that the genotoxic effect of DXR and various analogs thereof is uncoupled from the effect on GAr-mediated translation control. This is further supported by the observation that etoposide and teniposide, representing another class of topoisomerase-II–DNA targeting drugs, have no effect on GAr-mediated translation control. DXR and active analogs stimulate, in a GAr-dependent manner, EBNA1 expression in mammalian cells and overcome GAr-dependent restriction of MHC class I antigen presentation. These results validate our approach as an effective high-throughput screening assay to identify drugs that interfere with EBV immune evasion and, thus, constitute candidates for treating EBV-related diseases, in particular EBV-associated cancers.

Novel cationic bis(acylhydrazones) as modulators of Epstein–Barr virus immune evasion acting through disruption of interaction between nucleolin and G-quadruplexes of EBNA1 mRNA

“…Along these lines, we have previously developed a yeast (Saccharomyces cerevisiae)-based assay reproducing all the aspects of the GAr-based inhibition of translation, including the GAr-length dependency [18]. This allowed us to decipher the mechanisms of GAr-mediated mRNA translation suppression in cis and, in particular, to identify the cellular factors involved [19,20].…”

The long‐lasting love affair between the budding yeast Saccharomyces cerevisiae and the Epstein‐Barr virus

“…The ADE2 gene expressed from the constitutive ADH promoter, that allows its minimal expression to get white colonies, was fused to GAr domains of different lengths (21, 43 or 235 amino acids), or to the GAr domain of the Papio herpesvirus, which is known to have no effect on translation due to the insertion of one serine every seven residues of the repeat. As in human cells, the length‐dependent ability of the GAr domain to inhibit the translation of its own mRNA in cis was demonstrated whereas the Papio GAr domain had no effect [57]. Thanks to the Ade2p reporter protein, the effect of GAr can easily be monitored (Fig.…”

“…4C), but also to overcome the GAr‐dependent restriction of MHC class I antigen presentation (Fig. 4D) [57]. In addition, as many chemical analogs of DXR are commercially available, a structure‐activity relationship study was performed using both the yeast and T‐cell assays.…”

“…The good correlation between the results obtained using the two assays definitively validated the yeast model for EBNA1‐based EBV's stealthiness. They also indicated that the effect of DXR on the GAr‐mediated inhibition of translation is uncoupled from its already known genotoxic effect [57]. What's next?…”

Chémobiologie à l’happy hour

“…Comme attendu, la DXR augmente également la présentation antigénique qui était limitée par l'effet du domaine GAr sur la traduction. Tous ces résultats valident notre modèle levure d'analyse de la furtivité d'EBV [36,37]. La DXR est génotoxique (c'est d'ailleurs la base de son utilisation comme anticancéreux) et sa synthèse est complexe.…”