N ⁶-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection

Abstract

Methylation at the N ⁶ position of adenosine (m ⁶A) 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 m ⁶A deposition and proteins involved in the m ⁶A pathway play a diverse set of roles in either restricting or modulating the lifecycles of select viruses. Here, we report that m ⁶A levels are significantly increased in cells infected with the oncogenic human DNA virus Kaposi’s sarcoma-associated herpesvirus (KSHV). Transcriptome-wide m ⁶A-sequencing of the KSHV-positive renal carcinoma cell line iSLK.219 during lytic reactivation revealed the presence of m ⁶A across multiple kinetic classes of viral transcripts, and a concomitant decrease in m ⁶A levels across much of the host transcriptome. However, we found that depletion of the m ⁶A 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 m ⁶A 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 m ⁶A modified. In contrast, although the ORF50 mRNA was also m ⁶A 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 m ⁶A 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.

Author summary

In addition to its roles in regulating cellular RNA fate, methylation at the N ⁶ position of adenosine (m ⁶A) of mRNA has recently emerged as a mechanism for regulating viral infection. While it has been known for over 40 years that the mRNA of nuclear replicating DNA viruses contain m ⁶A, only recently have studies began to examine the distribution of this modification across viral transcripts, as well as characterize its functional impact upon viral lifecycles. Here, we apply m ⁶A-sequencing to map the location of m ⁶A modifications throughout the transcriptome of the oncogenic human DNA virus Kaposi’s sarcoma-associated herpesvirus (KSHV). We show that the m ⁶A machinery functions in a cell type specific manner to either promote or inhibit KSHV gene expression. Thus, the KSHV lifecycle is impacted by the m ⁶A pathway, but the functional outcome may depend on cell lineage specific differences in m ⁶A-based regulation.