RNA m⁶ A methylation regulates virus-host interaction and EBNA2 expression during Epstein-Barr virus infection

Introduction: N⁶ -methyladenosine (m⁶ A) is the most prevalent modification that occurs in messenger RNA (mRNA), affecting mRNA splicing, translation, and stability. This modification is reversible, and its related biological functions are mediated by "writers," "erasers," and "readers." The field of viral epitranscriptomics and the role of m⁶ A modification in virus-host interaction have attracted much attention recently. When Epstein-Barr virus (EBV) infects a human B lymphocyte, it goes through three phases: the pre-latent phase, latent phase, and lytic phase. Little is known about the viral and cellular m⁶ A epitranscriptomes in EBV infection, especially in the pre-latent phase during de novo infection.

Methods: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-RT-qPCR were used to determine the m⁶ A-modified transcripts during de novo EBV infection. RIP assay was used to confirm the binding of EBNA2 and m⁶ A readers. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and Western blot analysis were performed to test the effect of m⁶ A on the host and viral gene expression.

Results: Here, we provided mechanistic insights by examining the viral and cellular m⁶ A epitranscriptomes during de novo EBV infection, which is in the pre-latent phase. EBV EBNA2 and BHRF1 were highly m⁶ A-modified upon EBV infection. Knockdown of METTL3 (a "writer") decreased EBNA2 expression levels. The emergent m⁶ A modifications induced by EBV infection preferentially distributed in 3' untranslated regions of cellular transcripts, while the lost m⁶ A modifications induced by EBV infection preferentially distributed in coding sequence regions of mRNAs. EBV infection could influence the host cellular m⁶ A epitranscriptome.

Conclusions: These results reveal the critical role of m⁶ A modification in the process of de novo EBV infection.