UC Berkeley

More than 100 types of RNA modifications are known to exist, regulating key aspects of cellular biology and metabolism. First discovered to be present on viral RNAs in the 1970s, methylation at the N6 position of adenosine (m6A) is the most abundant internal modification within eukaryotic mRNAs, and is proposed to be dynamically regulated during times of cellular stress. However, the transcriptome-wide distribution and function of m6A in the lifecycle of a dsDNA virus had not been explored. In Chapter 1, I review how human herpesviruses manipulate host pathways in order to replicate, with particular emphasis on gammaherpesviruses. I also discuss the diverse roles played by RNA modifications in gene expression, focusing on enzymes that install m6A (writers), and proteins that bind m6A modified mRNA (readers). In Chapter 2, I present a detailed investigation of how the m6A pathway impacts the lifecycle of the oncogenic human virus Kaposi’s sarcoma-associated herpesvirus (KSHV). Mass spectrometry and transcriptome-wide m6A-sequencing revealed enrichment of m6A in viral transcripts upon lytic reactivation, including in the lytic transactivator ORF50. Depletion of the writer METTL3 and reader YTHDF2 in three different KSHV reactivation models had differential pro- and anti-viral impacts on viral gene expression depending on the cell-type analyzed. Finally, in Chapter 3, I put my findings in the broader context of the literature examining m6A modifications in viral lifecycles. I discuss technical challenges that have hindered research to-date, and potential reasons for the diverse pro and anti-viral effects ascribed to the m6A pathway, including a discussion of why the m6A pathway may have cell-type specific effects. I highlight the impact of m6A deposition on host transcripts, including those of the innate immune system. Overall, this study demonstrates the critical importance of m6A modifications in regulating KSHV lytic gene expression and sets the stage for future studies to elucidate more mechanistic details, including the potential for discrete impacts at different stages of the viral lifecycle.