Genome Persistence of KSHV

描述（由申请人提供）： 卡波西肉瘤相关疱疹病毒（Kaposi's sarcoma associated herpes virus，KSHV/HHV-8）是第二种被鉴定的与至少3种主要增殖性疾病相关的γ疱疹病毒。这些包括卡波西肉瘤和基于体腔的淋巴瘤（BCBL）或胸膜渗出性淋巴瘤（PEL）。 潜伏相关核抗原（拉娜）是主要的潜伏抗原之一， 在所有潜伏感染的KSHV细胞中组成型表达，并参与dsDNA KSHV基因组的长期稳定维持以及转录调控。 研究还表明，在调节病毒潜伏期中的重要作用，与病毒的直接感染有关。 早期反式激活因子Rta。拉娜与KSHV末端重复序列（TR）内的顺式作用元件结合，并与许多染色体蛋白质相关，这些蛋白质对于将病毒蛋白质束缚在一起以及将病毒基因组分离到新的子细胞中是重要的。 此外，当在体外人细胞中表达时，拉娜可诱导染色体畸变，表明拉娜在KSHV感染细胞中诱导肿瘤发生中的作用。 这项新提议的具体目的是集中我们的研究调查KSHV潜伏感染，通过病毒基因组的分离的持久性和机制，从亲本细胞传递到潜伏感染的后代细胞。目前还没有直接的研究全面描述了这种情况发生的具体机制。 我们已经证明了拉娜与细胞蛋白的关联，这些蛋白是感染细胞中病毒基因组的束缚和分离所必需的。 我们还显示了与核有丝分裂装置蛋白NuMA以及Bub 1和一些CenP蛋白的关联。 在这里，我们将探索与拉娜和与着丝粒和动粒相关的细胞蛋白质的直接联系，使用现有技术的体内可视化策略结合靶向的shRNA敲除特定的细胞靶点，为了了解这些蛋白质在介导或控制KSHV基因组DNA从一个子细胞传递到另一个子细胞时的过渡状态中的功能，其次是不丢失基因组。我们将使用直接的生物化学、遗传学和体内FRET可视化分析来关注拉娜与动粒蛋白Bub 1和已知调节该蛋白复合物稳定性的APC蛋白的相互作用，并研究这种蛋白复合物的稳定性机制，从而使拉娜和KSHV基因组能够在有丝分裂阶段进行而不损失其蛋白质。因此，病毒基因组可以分离到新的子细胞中。

DESCRIPTION (provided by applicant): Kaposi's sarcoma associated herpes virus (KSHV/HHV-8) is the second identified gammaherpesvirus associated with at least 3 major proliferative diseases. These include Kaposi's sarcoma and body cavity based lymphomas (BCBLs) or Pleural Effusion Lymphomas (PELs). One of the major latent antigens, latency- associated nuclear antigen (LANA) is constitutively expressed in all latently infected KSHV cells and is involved in the long erm maintenance of the dsDNA KSHV genome as well as transcriptional regulation. Studies have also shown an important role in regulation of viral latency in association with the viral immediate early transactivator Rta. LANA binds to cis-acting elements within the KSHV terminal repeats (TR) and is associated with a number of chromosomal proteins which are important for tethering the viral proteins and segregation of the viral genome to new daughter cells. Furthermore, LANA can induce chromosomal abberations when expressed in human cells in vitro suggesting a role for LANA in induction of oncogenesis in KSHV infected cells. The specific aims of this new proposal is to focus our studies investigating KSHV latent infection, through persistence and mechanism of segregation of the viral genome which is passed from parental cell to progeny cells which are latently infected. There has been no direct studies which comprehensively describes a specific mechanism by which this occurs. We have shown associations of LANA with cellular proteins that are necessary for tethering and segregation of the viral genome in the infected cells. We have also shown an association with the nuclear mitosis apparatus protein NuMA, as well as Bub1 and a number of CenP proteins. Here we will explore the direct connections with LANA and cellular proteins associated with the centromere and kinetochore using state of the art in vivo visualizatin strategies combined with targeted shRNA knockdown of specific cellular targets, to understand the function of these proteins in mediating or controlling the transitional states of the KSHV genomic DNA as it passes from one daughter cell to the next without loss of the genome. We will use direct biochemical, genetics and in vivo FRET visualization analyses to focus on the interaction of LANA with the kinetochore protein Bub1 and the APC proteins known to regulate the stability of this protein complex, and to investigate the mechanism of stability of this protein complex whereby LANA and the KSHV genome can progress through the stages of mitosis without loss of the genome thus allowing segregation of the viral genomes to the new daughter cells.