Kaposi's sarcoma-associated herpesvirus (KSHV) genomic episomes encode nuclear non-coding RNAs, including PAN RNA. This non-coding RNA is essential for KSHV gene expression and replication during de novo infection. Although the biological significance of the non-coding RNA has been well documented, how the nuclear non-coding RNA functions in this role remains elusive. Recent sequencing studies showed that although the genomic DNA sequence of non-coding RNAs are not conserved, the positions are well-conserved among different species. Further studies demonstrated that deletion of non-coding RNA expression diminished expression of surrounding coding genes. Based upon these facts, we formulated the hypothesis that one of the functions of viral long-non-coding RNA is to be transcribed at the genomic region at higher frequencies during the course of viral replication. Our preliminary studies demonstrated that KSHV PAN RNA expression is maintained by two immediate-early proteins, and expression of the PAN RNA leads to activation of downstream viral ORFs in reporter assays. In this application, we aim to dissect the interactions between two immediate-early proteins and a viral nuclear non-coding RNA (PAN RNA). Specifically, we intend to explore the model in which two immediate- early proteins establish viral transcriptional centers though expression of non-coding RNA. We will study the model that the genomic locus encoding a long non-coding RNA physically traps cellular RNA polymerase II via formation of local genomic loops and locally concentrates transcriptional machinery; this mechanism allows the KSHV genome to be robustly and preferentially expressed in the cellular 3D nuclear space. Both genomic and biochemical approaches will be utilized to clarify the function of the viral non-coding RNA. We also believe that inhibition of interactions to assemble transcriptional center will disrupt KSHV gene expression, and consequently replication. This application will reveal new aspects of the spatio-temporal (4D) organization of the cell nucleome by using the KSHV mini-episome as a well-defined model for chromosomal dynamics.