Spatiotemporal Gene Regulation and KSHV Replication

卡波济肉瘤相关疱疹病毒（KSHV）基因组游离体在三个细胞中动态移动， 三维（3D）核空间，最终聚集在重新激活细胞。细胞RNA聚合酶II（RNA pol II）也易位到聚集的病毒基因组上，导致病毒转录因子的形成。 工厂这种以前未表征的机制代表了KSHV基因调控的另一层， 这有助于有限数量的细胞RNA pol II的再利用和再利用， 基因表达。了解这种机制可能使我们能够确定新的靶点来抑制病毒 复制的 在这个应用中，我们提出了一个全面的研究，以（1）确定关键的细胞蛋白质参与 与病毒转录工厂的结构或功能;（2）确定非编码PAN RNA转录本的作用 及其在病毒转录工厂形成中的基因组区域;以及（3）可视化病毒转录工厂的物理组装。 使用活的3D荧光显微镜在重新激活的宿主细胞的细胞核中的转录工厂。我们将 还通过超分辨率光学成像技术表征了病毒转录工厂的分子结构 相关电子显微镜成像显微镜。通过这种多方面的做法，我们希望 了解病毒转录工厂在KSHV基因表达扩增中的作用， 这将导致开发新的治疗策略KSHV相关的恶性肿瘤，通过抑制病毒 复制的此外，我们希望这项调查将阐明时空（4D）组织的 细胞核组：使用KSHV作为一个明确的模型染色体，我们可以很容易地跟踪宿主细胞 细胞核的生物化学和遗传学方法。

Kaposi's sarcoma-associated herpesvirus (KSHV) genomic episomes dynamically move in three- dimensional (3D) nuclear space to ultimately aggregate in reactivating cells. Cellular RNA polymerase II (RNA pol II) also translocates onto the aggregated viral genomes, resulting in the formation of viral transcriptional factories. This previously uncharacterized mechanism represents an additional layer of KSHV gene regulation, which facilitates the reutilization and repurposing of the limited quantities of cellular RNA pol II for effective viral gene expression. Understanding this mechanism may allow us to identify new targets to inhibit viral replication. In this application, we propose a comprehensive study to (1) identify the key cellular proteins involved with viral transcriptional factory structure or function; (2) determine the role of noncoding PAN RNA transcripts and its genomic region in viral transcriptional factory formation; and (3) visualize the physical assembly of viral transcriptional factories in the nuclei of reactivated host cells using live 3D fluorescence microscopy. We will also characterize the molecular architecture of viral transcriptional factories by super-resolution optical microscopy with correlative electron microscopic imaging. Through this multifaceted approach, we hope to understand the role of viral transcriptional factories in the amplification of KSHV gene expression, which may in turn lead to the development of new therapeutic strategies for KSHV-associated malignancies by inhibiting viral replication. In addition, we expect this investigation will shed light on the spatio-temporal (4D) organization of the cell nucleome: using KSHV as a well-defined model chromosome that we can readily track in the host cell nucleus with biochemical and genetic approaches.