Genome Persistence of KSHV

描述(由申请人提供)： 卡波西肉瘤相关疱疹病毒(KSHV/HHV-8)是第二种已鉴定的与至少3种主要增生性疾病有关的伽马疱疹病毒。这些包括卡波西肉瘤和体腔淋巴瘤(BCBL)或胸腔积液淋巴瘤(PEL)。潜伏相关核抗原(LANA)是一种主要的潜伏抗原 在所有潜伏感染的KSHV细胞中结构性表达，并参与dsDNA KSHV基因组的长期ERM维持以及转录调控。研究还表明，在调节病毒潜伏期方面，与病毒即刻有关 早期反式激活因子RTA。LANA与KSHV末端重复序列(TR)中的顺式作用元件结合，并与许多染色体蛋白有关，这些蛋白对捆绑病毒蛋白和将病毒基因组分离到新的子细胞非常重要。此外，当LANA在体外表达于人类细胞中时，可以诱导染色体的振荡，这表明LANA在诱导KSHV感染细胞的肿瘤形成中发挥了作用。这一新建议的具体目的是通过病毒基因组从亲代细胞传递到潜伏感染的子代细胞的持久性和分离机制，重点研究KSHV的潜伏感染。目前还没有直接的研究来全面描述这种情况发生的具体机制。我们已经展示了LANA与细胞蛋白的关联，这些蛋白是在感染细胞中拴系和分离病毒基因组所必需的。我们还显示了与核有丝分裂装置蛋白NUMA，以及Bub1和一些CENP蛋白的关联。在这里，我们将探索与LANA和与着丝粒和着丝粒相关的细胞蛋白质的直接联系，使用最先进的体内可视化策略结合特定细胞靶点的靶向shRNA敲除，以了解这些蛋白质在调节或控制KSHV基因组DNA从一个子细胞传递到下一个细胞时的过渡状态而不损失基因组。我们将使用直接的生化、遗传学和体内FRET可视化分析来关注LANA与动粒蛋白Bub1和APC蛋白之间的相互作用，并研究这种蛋白质复合体的稳定性机制，通过这种稳定的机制，LANA和KSHV基因组可以在不损失基因组的情况下通过有丝分裂阶段，从而允许病毒基因组分离到新的子细胞。

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.