Human herpesvirus 8 (HHV-8 ) is associated with a growing list of malignancies, especially in HIV-1 infected patients. HHV-8 is likely to be the etiologic agent of Kaposi's sarcoma. HHV-8 is also found in AIDS-associated B cell lymphomas: primary effusion lymphoma (PEL) and multicentric Castlemen's disease. Understanding the mechanism of HHV-8 replication is critical for the development of effective therapeutic strategies for HHV-8 related diseases and is the long-term goal of our research program. A novel non-coding transcript, polyadenylated nuclear (PAN) RNA has been discovered as an early lytic RNA of HHV-8 . In both primary effusion lymphoma and Kaposi's sarcoma tumor lesions, PAN RNA is transcribed in a subset of tumor cells in which the virus is undergoing lytic replication. PAN RNA has three striking features: (1) it is localized exclusively in the nuclear speckles, unlike normally polyadenylated RNAs which are transported to the cytoplasm; (2) it forms a RNP complex with cellular proteins, some of which are autoantigens; (3) it encompasses 80% of total poly (A)-plus RNAs in the cells. Therefore, PAN RNA represents a novel type of RNA. Our objective is to understand the structure and function of PAN RNA and its associated proteins. A combination of biochemical and genetic methods is proposed in this application to achieve the following specific aims: 1. Identification of the sequence elements controlling the transcription initiation of PAN RNA and the formation of the RNP complex which is localized in the nuclear speckles. 2. Determination of the RNA secondary structure in the native complex and identification of the protein components of the complex to unravel the cellular processes with which PAN RNA interacts. 3. Determination of the function of PAN RNA during viral lytic replication and the effects of PAN RNA expression on cellular processes.
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