Genetic and Biochemical Studies of the KSHV LANA Gene

描述（由申请方提供）：卡波西肉瘤（KS）相关疱疹病毒（KSHV）与KS、原发性渗出性淋巴瘤（PEL）和多中心Castleman病在病因学上相关。这些疾病发生在艾滋病和其他患者中，目前的治疗方法有限。KSHV潜伏感染绝大多数肿瘤细胞，并且病毒DMA作为多拷贝、染色体外、环状附加体持续存在。为了在增殖细胞中持续存在，附加体必须复制并有效地分离到子核。KSHV潜伏期相关核抗原（拉娜）结合末端重复序列（TR）DNA介导KSHV附加体持久性。拉娜介导TR DMA复制，并将附加体拴系到有丝分裂染色体上，以有效分离到后代细胞核中。 在我们对拉娜介导的附加体维持的理解中仍然存在重大差距。拉娜染色体联合对于附加体系留是必不可少的，然而染色体附着的潜在机制尚不清楚。拉娜是一种大的多功能蛋白质。虽然拉娜与其他γ-2疱疹病毒的拉娜同源物具有序列同源性和附加体维持功能，但它比其他拉娜同源物大得多。附加体维持所需的拉娜结构域的定义对于理解KSHV附加体持久性至关重要。拉娜介导的DNA复制和附加体维持的精确顺式作用TR序列要求是未知的。由于TR占KSHV基因组的约20%，因此理解必要的顺式作用附加体持久序列对于更好地理解KSHV生物学是至关重要的。 这项工作将调查拉娜的附加体维护功能的关键方面。拉娜N-和C-末端独立地与染色体结合的机制将被阐明。将鉴定附加体持久性所需的拉娜结构域。将定义拉娜介导的附加体持久性的最小顺式作用TR序列要求。由于附加体持续性对于潜伏感染是至关重要的，并且大多数肿瘤细胞是潜伏感染的，因此这项工作可能导致预防和治疗KSHV相关恶性肿瘤的新策略。此外，附加体维持所需的拉娜和顺式作用元件的定义将增强基于拉娜的基因治疗载体的构建。

DESCRIPTION (provided by applicant): Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is etiologically linked with KS, primary effusion lymphomas (PELs) and multicentric Castleman's disease. These diseases occur in AIDS and other patients and current therapies are limited. KSHV latently infects the vast majority of tumor cells and viral DMA persists as a multiple copy, extrachromosomal, circular episome. To persist in proliferating cells, episomes must replicate and efficiently segregate to daughter nuclei. KSHV latency associated nuclear antigen (LANA) binds terminal repeat (TR) DNA to mediate KSHV episome persistence. LANA mediates TR DMA replication and tethers episomes to mitotic chromosomes for efficient segregation to progeny nuclei. Significant gaps remain in our understanding of LANA mediated episome maintenance. LANA chromosome association is essential for episome tethering yet the mechanisms underlying chromosome attachment are ill-defined. LANA is a large, multifunctional protein. Although LANA shares sequence homology and episome maintenance function with LANA homologs of other gamma-2 herpesviruses, it is much larger than the other LANA homologs. Definition of the LANA domains required for episome maintenance is central to understanding KSHV episome persistence. The precise cis-acting TR sequence requirements for LANA mediated DNA replication and episome maintanence are unknown. Since TRs account for -20% of the KSHV genome, an understanding of the necessary cis-acting episome persistence sequence is fundamental to a better understanding of KSHV biology. This work will investigate critical aspects of LANA's episome maintenance function. The mechanisms by which the LANA N- and C-termini independently associate with chromosomes will be elucidated. The LANA domains required for episome persistence will be identified. The minimal cis-acting TR sequence requirements for LANA mediated episome persistence will be defined. Since episome persistence is critical for latent infection and most tumor cells are latently infected, this work may lead to new strategies for prevention and treatment of KSHV associated malignancies. Further, definition of the LANA and cis-acting element requirements for episome maintenance will enhance construction of LANA based gene therapy vectors.