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

BackgroundReactivation of Epstein Barr virus (EBV) leads to modulation of the viral and cellular epitranscriptome. N6-methyladenosine (m(6)A) modification is a type of RNA modification that regulates metabolism of mRNAs. Previous reports demonstrated that m(6)A 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.MethodsMethylated 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 m(6)A methylation sites was used to verify the effect of m(6)A methylation on its stability and expression levels.ResultsWe report that during EBV reactivation the m(6)A 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 m(6)A 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.ConclusionOur findings illuminate the significance of m(6)A 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.

Automated summary

This study reveals that during EBV reactivation, the downregulation of m(6)A eraser ALKBH5 leads to enhanced methylation of cellular transcripts DTX4 and TYK2, resulting in degradation of TYK2 mRNA and increased translation efficiency of DTX4 mRNA. This attenuates the IFN signaling, promoting viral lytic replication.