Regulation of KSHV replication by N6-methyladenosine (m6A) - Diversity Supplement

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N6-methyladenosine (m6A) is the most abundant internal modification on poly(A) RNA. Dynamic regulation of the m6A epitranscriptome is involved in diverse cellular functions. m6A mediates these functions by affecting mRNA translation, alternative splicing, nuclear export and degradation, and miRNA biogenesis and binding through three groups of proteins: methyltransferases or “writers”, demethylases or “erasers”, and m6A-binding proteins or “readers”. m6A is present in the genomes of RNA viruses and regulates the replication of these viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) commonly found in AIDS patients. Understanding the mechanism regulating KSHV latent and lytic replication can not only provide insights into the pathogenesis of KSHV-induced cancers but also serve as the basis for developing novel therapy. In the current funding period, we have made significant progresses toward this goal. For this renewal, we have discovered that m6A is abundant on KSHV transcripts, and that m6A reader protein YTHDF2 acts as an antiviral factor during viral lytic replication. We demonstrate m6A dynamics during KSHV latent and lytic replications, and in different cell types that support distinct viral replication programs. Despite these works, the roles of m6A in KSHV infection have just started to be revealed. The Objective of this application is to systematically map the dynamics of m6A modifications at a single base resolution and bindings of m6A reader proteins in KSHV epitranscriptome, and determine their functions in different phases of KSHV life cycle, and KSHV-induced tumorigenesis. The Central Hypothesis is that KSHV m6A modifications are dynamically regulated, and these modifications mediate different phases of KSHV life cycle, and hence KSHV-induced tumorigenesis. We will test this hypothesis by mapping m6A marks in KSHV transcriptome at a single base resolution, and determine the roles of m6A writer and eraser proteins in different phases of KSHV life cycle and KSHV-induced tumorigenesis (Aim 1); determining the functions of m6A reader proteins in different phases of KSHV life cycle by gain- and loss-of-function approaches and by examining their bindings to KSHV transcripts (Aim 2); and examining the functions of KSHV m6A marks in the context of viral infection using Crispr-Cas9-guided m6A writer and eraser, and by site-specific mutagenesis (Aim 3). It is our expectations that this project will provide comprehensive mapping and functional delineation of m6A marks, and m6A writer, eraser and reader proteins in different phases of KSHV life cycle, and KSHV- induced tumorigenesis. This work is highly significant as it will, for the first time, systematically reveal the functions of these RNA modifications in KSHV infection, thus providing insights into the mechanism regulating KSHV life cycle and KSHV-induced pathogenesis. This study will also identify potential prognostic markers and therapeutic targets. The proposed work is highly innovative as it will use new technologies to map m6A marks and bindings of m6A reader proteins, as well as directly manipulate m6A marks on key viral transcripts by Crispr-Cas9-guided m6A writer and eraser, KSHV reverse genetics, and innovative tumor models. Furthermore, the generated datasets, information and reagents will be valuable to the scientific community.