N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection

Methylation at the N⁶ position of adenosine (m⁶A) is a highly prevalent and reversible modification within eukaryotic mRNAs that has been linked to many stages of RNA processing and fate. Recent studies suggest that m⁶A deposition and proteins involved in the m⁶A pathway play a diverse set of roles in either restricting or modulating the lifecycles of select viruses. Here, we report that m⁶A levels are significantly increased in cells infected with the oncogenic human DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV). Transcriptome-wide m⁶A-sequencing of the KSHV-positive renal carcinoma cell line iSLK.219 during lytic reactivation revealed the presence of m⁶A across multiple kinetic classes of viral transcripts, and a concomitant decrease in m⁶A levels across much of the host transcriptome. However, we found that depletion of the m⁶A machinery had differential pro- and anti-viral impacts on viral gene expression depending on the cell-type analyzed. In iSLK.219 and iSLK.BAC16 cells the pathway functioned in a pro-viral manner, as depletion of the m⁶A writer METTL3 and the reader YTHDF2 significantly impaired virion production. In iSLK.219 cells the defect was linked to their roles in the post-transcriptional accumulation of the major viral lytic transactivator ORF50, which is m⁶A modified. In contrast, although the ORF50 mRNA was also m⁶A modified in KSHV infected B cells, ORF50 protein expression was instead increased upon depletion of METTL3, or, to a lesser extent, YTHDF2. These results highlight that the m⁶A pathway is centrally involved in regulating KSHV gene expression, and underscore how the outcome of this dynamically regulated modification can vary significantly between cell types.