Genetic and Biochemical Studies of KSHV LANA

KSHV LANA 的遗传和生化研究

• 批准号: 9031719
• 负责人: Kenneth M Kaye
• 金额: $ 42.16万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9031719
• 关键词: Acquired Immunodeficiency Syndrome; Africa South of the Sahara; Atomic Force Microscopy; Behavior; Binding; Binding Sites; Biochemical; Biological; Biology; C-terminal; Cell Nucleus; Cell Survival; Cell division; Cells; Chromatin Loop; Chromosomes; Collaborations; Complement; Complex; DNA; DNA Binding; DNA Binding Domain; Daughter; Development; Electron Microscopy; Epidemic; Episome; Future; Genetic; Health; Herpesviridae; Herpesviridae Infections; Histone H2A; Human; Human Herpesvirus 8; Immune; Investigation; Kaposi Sarcoma; Laboratories; Latent virus infection phase; Lead; Link; Malignant Neoplasms; Mediating; Mitosis; Mitotic Chromosome; Multicentric Angiofollicular Lymphoid Hyperplasia; Mutate; N-terminal; Nucleosomes; Oral cavity; Organ; Plasmids; Preventive; Process; Proliferating; Role; Sister Chromatid; Site; Structure; Surface; Terminal Repeat Sequences; Therapeutic; Viral; Viral Genome; Virus; Virus Latency; Visceral; Work; X-Ray Crystallography; antigen binding; daughter cell; insight; latency-associated nuclear antigen; live cell imaging; live cell microscopy; mutant; neoplastic cell; novel; prevent; primary effusion lymphoma; segregation; tumor

 DESCRIPTION (provided by applicant): Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the etiologic agent of KS, primary effusion lymphoma (PEL) and multicentric Castleman's disease. These tumors occur in AIDS and other immune compromised states and current therapies are limited. KS is the leading AIDS malignancy, is epidemic in sub Saharan Africa, and often involves the oral cavity and visceral organs. KSHV latently infects tumor cells and viral genomes persists as multiple copy, extrachromosomal, circular episomes. In order to persist in dividing cells, episomes must replicate and efficiently segregate to daughter nuclei. The latency-associated nuclear antigen (LANA) mediates KSHV episome persistence and is essential for latency and virus survival. There are two components to episome persistence: 1) replication of KSHV DNA and 2) segregation of replicated episomes to daughter cell nuclei. To segregate episomes to daughter nuclei, LANA tethers KSHV terminal repeat (TR) DNA to mitotic chromosomes. The tethering occurs through the C- terminal LANA DNA binding domain (DBD) binding TR DNA and N-terminal LANA simultaneously binding histones H2A/H2B on the nucleosome surface. In addition, we recently discovered an internal LANA sequence critical for segregation that does not bind DNA or chromosomes, suggesting it is likely involved in aspects of the segregation process that remain to be elucidated. LANA's ability to segregate episomes is essential to KSHV latency, yet little is known regarding the mechanisms underlying this process. The process of segregation is likely complex, with important questions unanswered. For instance, how does the recently discovered internal LANA sequence contribute to segregation? During mitosis, does random episome attachment occur to chromosomes resulting in random distribution to daughter cells, or is there tight regulatory control targeting each replicated episome to a different sister chromatid for equal distribution to daughter cells? Fundamental questions as to how LANA binds TR DNA to tether episomes to mitotic chromosomes are unanswered. An understanding of these questions is critical to gaining insight into this key aspect of KSHV biology. This proposal investigates the process of LANA mediated segregation of episomes to daughter cells, which is essential for latency and virus survival. Work will define and functionally characterize the recently identified critical internal LANA effector sequence for segregation. State of the art live cell microscopy will be used to investigate episome replication and segregation dynamics. Work will also solve the structure of LANA complexed with DNA, which is central to an understanding of LANA's tethering mechanism. This work will provide comprehensive insight into LANA's essential segregation function and potentially lead to strategies that prevent or treat KSHV associated malignancies.

 描述（由适用提供）：Kaposi的肉瘤（KS）相关疱疹病毒（KSHV）是KS，一级积液淋巴瘤（PEL）和多中心骑士病的病因学药。这些肿瘤发生在艾滋病和其他免疫组合态中，当前疗法受到限制。 KS是领先的艾滋病恶性肿瘤，在撒哈拉以南非洲是流行病，通常涉及口腔和内脏器官。 KSHV潜在地感染的肿瘤细胞和病毒基因组持续为多个拷贝，外染色体，圆形发作。为了持续划分细胞，偶发必须复制并有效地分离为女儿核。潜伏期相关的核抗原（LANA）介导了KSHV偶发性持久性，对于潜伏期和病毒生存至关重要。沿着依赖性的持久性有两个组成部分：1）复制KSHV DNA和2）将复制的偶发分离为子细胞核心。为了将偶发分离为女儿核里，拉娜（Lana）tethers kshv末端重复（TR）DNA与有丝分裂染色体。束缚通过c-末端LANA DNA结合结构域（DBD）结合TR DNA和N末端LANA简单结合组蛋白H2A/H2B在核能表面上。此外，我们最近发现了一个至关重要的内部LANA序列，例如如何结合DNA或染色体，这表明它可能参与了隔离过程的各个方面，尚待阐明。拉娜（Lana）隔离插花对于KSHV潜伏期至关重要，但对于此过程的基础机制知之甚少。隔离过程可能很复杂，并且没有解决重要问题。例如，最近发现的内部LANA序列如何促进隔离？在有丝分裂期间，是否会随机发作附着在染色体上，导致随机分布到子细胞上，或者是否存在针对每个复制的发作的严格的调节控制，将每个复制的发作靶向不同的姐妹染色单体，以相等地分布到子细胞中？关于LANA如何将TR DNA与链球偶发与有丝分裂染色体结合的基本问题未得到解答。对这些问题的理解对于了解KSHV生物学的这一关键方面至关重要。该提案调查了LANA介导的偶发分离对子细胞的过程，这对于潜伏期和病毒生存至关重要。工作将定义并在功能上表征最近确定的隔离的关键内部LANA效应子序列。最先进的现场细胞显微镜将用于研究偶发复制和隔离动力学。工作还将解决与DNA复合的LANA结构，这对于理解Lana的束缚机制至关重要。这项工作将为Lana的基本隔离功能提供全面的见解，并有可能导致预防或治疗KSHV相关的恶性肿瘤的策略。