Attenuation of IFN signaling due to m6A modification of the host epitranscriptome promotes EBV lytic reactivation

Abstract

Background: Reactivation of Epstein Barr virus (EBV) leads to modulation of the viral and cellular epitranscriptome. N6-methyladenosine (m⁶A) modification is a type of RNA modification that regulates metabolism of mRNAs. Previous reports demonstrated that m⁶A modification affects the stability and metabolism of EBV encoded mRNAs. However, the effect of reactivation on reprograming of the cellular mRNAs, and how this contributes to successful induction of lytic reactivation is not known.

Methods: Methylated RNA immunoprecipitation sequencing (MeRIP-seq), transcriptomic RNA sequencing (RNA-seq) and RNA pull-down PCR were used to screen and validate differentially methylated targets. Western blotting, quantitative real-time PCR (RT-qPCR) and immunocytochemistry were used to investigate the expression and localization of different proteins. RNA stability and polysome analysis assays were used to detect the half-lives and translation efficiencies of downstream genes. Insertion of point mutation to disrupt the m⁶A methylation sites was used to verify the effect of m⁶A methylation on its stability and expression levels.

Results: We report that during EBV reactivation the m⁶A eraser ALKBH5 is significantly downregulated leading to enhanced methylation of the cellular transcripts DTX4 and TYK2, that results in degradation of TYK2 mRNAs and higher efficiency of translation of DTX4 mRNAs. This resulted in attenuation of IFN signaling that promoted progression of viral lytic replication. Furthermore, inhibition of m⁶A methylation of these transcripts led to increased production of IFN, and a substantial reduction in viral copy number, which suggests abrogation of lytic viral replication.

Conclusion: Our findings illuminate the significance of m⁶A modification in overcoming the innate immune response during EBV reactivation. We now report that during lytic reactivation EBV targets the RNA methylation system of the host to attenuate the innate immune response by suppressing the interferon signaling which facilitates successful lytic replication of the virus.

Keywords: Epitranscriptome; Innate Immune response; Lytic reactivation; N6-methyladenosine; RNA immunoprecipitation.