This proposal will shed light on the m6A epitranscriptomic regulation of polyadenylated nuclear (PAN) long non-coding (lnc) RNA in the context of Kaposi's sarcoma-associated herpesvirus (KSHV) replication. KSHV encodes a plethora of products to govern its two phases of infection, but also to intricately subvert normal cellular pathways, and establish a lifelong infection. During the latency, KSHV exists in a dormant state, and even though PAN lncRNA expression has been reported, its function and potential epitranscriptomic regulation have not been determined. KSHV lytic reactivation occurs in a highly defined order, with PAN being one of the most abundantly expressed transcripts. Numerous studies designate the critical function of PAN in regulating the cellular and viral gene expression, and export of viral RNAs out of the nucleus, yet, PAN interactome and its regulation have not been explored. Epitranscriptomic modifications, with m6A being the most prevalent, have been shown to affect RNA stability, structure, interactions, and play critical roles in the lifecycle of many human pathogenic viruses . In this proposal, we will explore the role of m6A on PAN RNA biology, and the influence of PAN m6A status on the KSHV gene expression program, and virion production. In Aim 1: we will define the PAN m6A localization and related methylome during KSHV replication. Our preliminary data show the precise localization of m6A on PAN during KSHV lytic reactivation and indicate specific m6A-related cellular enzymes, as PAN interacting partners. Using siRNAs, we will knockdown these enzymes to verify their involvement in PAN modification. The in vitro methyltransferase and demethylase assays will provide insight into the kinetics of the process, and the site-directed mutagenesis will allow delineating the RNA structural determinants that facilitate m6A deposition on PAN In Aim 2: we will explore the influence of m6A on PAN RNA biology and KSHV replication. We have identified the impact of m6A on PAN RNA structure by performing the SHAPE-MaP probing in BCBL-1 cells with a siRNA-directed knockdown of two major enzymes that regulate m6A. We have also established the bacterial artificial chromosome system that reconstitutes infectious KSHV in HEK293, and provides a tractable tool, allowing the direct manipulation of m6A sites. This system will be employed to address 1) the role of m6A in mediating PAN intermolecular contacts by applying the established in our lab RNA antisense purification and mass-spectrometry protocols, 2) investigate how the disruption of m6A on PAN affects its stability and subcellular localization, and 4) the influence of PAN m6A status over the KSHV gene expression program. The results of this proposal are expected to provide a better understanding of `epitranscriptomic code' of lncRNA that can apply to a much larger non-coding RNA population, provide the first insight in the scarcely addressed relationship between the viral lncRNAs and cellular epitranscriptomic processes, and identify the influence of PAN m6A modification over KSHV lifecycle.