Project Summary/Abstract The roles of noncoding (nc)RNAs in lymphoid cells harboring each of three oncogenic herpesviruses are being investigated. Epstein-Barr virus (EBV) infects and transforms human B cells; it is the causative agent of infectious mononucleosis and is associated with several human cancers. Herpesvirus saimiri (HVS) induces fatal lymphomas and leukemias in New World monkeys and transforms human T lymphocytes in culture. Kaposi's sarcoma-associated herpesvirus (KSHV) afflicts immunocompromised individuals and persists in a latent form until lytic activation. In recent years, we have focused our efforts on the structure and functions of the two EBV-encoded EBERs, the seven HVS-encoded HSURs and six HVS microRNAs, as well as the KSHV PAN RNA. These viral ncRNAs are all abundant, conserved between related viruses and bind host proteins to form ncRNPs. Our functional studies have uncovered novel mechanisms of microRNA biogenesis and decay, revealed that viral ncRNPs can be essential for nuclear processes as diverse as viral DNA replication (EBER2) or mRNA export to the cytoplasm (PAN), identified the role of triple helices in RNA stabilization, and contributed important insights into viral evolution. Most compelling is that our studies of viral ncRNAs have uncovered the existence of and begun to elucidate novel cellular mechanisms such as the regulation of cellular microRNA populations and how the polyA tail and 3′UTR may collaborate to stabilize cellular mRNAs. Proposed aims will exploit these advances to further investigate the underlying molecular mechanisms. We shall extend our original discovery of target-directed microRNA decay (TDMD) to identify proteins and additional RNA signals contributing to cellular microRNA degradation, as well as investigate the role of a putative small RNA derived from the SARS-CoV-2 genome in regulating host immune responses, with potential diagnostic/therapeutic implications. We shall establish how its polyA tail as well as internal sequences contribute to PAN RNA's ability to enable the nuclear export of late lytic mRNAs, leading to virion protein production and virion release. We shall search cellular transcriptome databases for the presence of RNA sequence/structure motifs contributing to polyA-3′UTR interactions (and presumably RNA stabilization), as recently revealed by our high-resolution X-ray analyses. Newly discovered triplex-forming elements (ENEs) in coronavirus RNAs will be analyzed for their stabilization activity and possible contributions to viral protein synthesis, with potential therapeutic applications. Extensive interactions between viral transcripts in EBV- infected cells discovered by psoralen crosslinking will be validated and further analyzed.

Project Narrative We are studying how viruses that cause lymphomas or sarcomas in humans or monkeys use RNA molecules to manipulate their host cells to the advantage of the virus. These RNAs are not templates for making proteins but act to alter how other genes function after infection. Understanding these viral noncoding RNAs will provide novel targets for the design of therapeutics to combat these cancer-causing viruses, as well as many insights into host-cell processes.