Genetic and Biochemical Studies of KSHV LANA

KSHV LANA 的遗传和生化研究

• 批准号: 10599894
• 负责人: Kenneth M Kaye
• 金额: $ 65.55万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10599894
• 关键词: Acquired Immunodeficiency Syndrome; Africa South of the Sahara; Animal Model; Binding; Biochemical; Biological; Biological Assay; C-terminal; Cell Nucleus; Cell Proliferation; Cell Survival; Cells; DNA; DNA Binding Domain; DNA biosynthesis; Daughter; Development; Disease; Elements; Epidemic; Episome; Etiology; Genes; Genetic; Genome; Glycogen Synthase Kinase 3; Herpesviridae Infections; Histone H2A; Human; Human Herpesvirus 8; Immunocompromised Host; Investigation; Kaposi Sarcoma; Link; Lysine; Maintenance; Malignant Neoplasms; Mediating; Mitotic Chromosome; Molecular; Multicentric Angiofollicular Lymphoid Hyperplasia; Mus; N-terminal; Nucleosomes; Oncoproteins; Oral cavity; Organ; Phosphorylation Site; Plasmids; Proliferating; Proteins; Reader; Repetitive Sequence; Role; Sequence Deletion; Sequence Homologs; Specificity; Surface; TP53 gene; Terminal Repeat Sequences; Viral; Viral Genome; Virus; Virus Replication; Visceral; Work; antigen binding; daughter cell; experimental study; gamma-2 herpesvirus; gammaherpesvirus; genetic approach; in vivo; insight; interest; latency-associated nuclear antigen; neoplastic cell; novel; novel strategies; prevent; primary effusion lymphoma; segregation; tumor; ubiquitin-protein ligase; viral DNA

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV or HHV8) is the causative agent of KS, primary effusion lymphoma (PEL) and multicentric Castleman's disease. These tumors occur in AIDS and other immunocompromised states. There are no specific therapies for these diseases. KS is the leading AIDS malignancy, and is epidemic in sub Saharan Africa. KS often involves the oral cavity and can disseminate to visceral organs. KSHV latently infects tumor cells and viral genomes persist as circular, multiple copy, extrachromosomal, episomes (plasmids). In order to persist in proliferating cells, episomes must first replicate and then efficiently segregate to daughter nuclei. Tumor cell viability is dependent on latent KSHV infection. The latency-associated nuclear antigen (LANA) mediates KSHV episome persistence and this function is essential for virus survival. Therefore, LANA function is of fundamental importance. LANA represents the Achilles heel of KSHV as its disruption is lethal to the virus. LANA is necessary and sufficient for KSHV episome maintenance in the absence of other virus genes. Episome persistence is comprised of two components: 1) replication of viral DNA and 2) segregation of the replicated episomes to progeny cell nuclei. LANA mediates both these functions. C-terminal LANA binds specific sequence in KSHV terminal repeat (TR) DNA to mediate DNA replication. LANA tethers TR DNA to mitotic chromosomes to segregate genomes to daughter nuclei. To form a molecular tether, the C-LANA DNA binding domain (DBD) binds TR DNA and N-LANA simultaneously binds histones H2A/H2B at the folded portion of the nucleosome. In addition to N- and C-LANA, internal LANA sequence is also critical for episome persistence. We have discovered two distinct internal LANA regions, one comprised of unique sequence, and one within repetitive sequence, that are critical for LANA mediated episome persistence. The unique sequence contains three distinct functional motifs, whereas the repetitive region is homologous to a host cell “reader” sequence. Both of these regions will be investigated in this work. This work will use a detailed, in depth approach to investigate each of the recently identified LANA functional sequences. Experiments will use a rigorously developed panel of assays to identify the functional motif and determine the mechanism through which the unique sequence mediates episome persistence. The repetitive LANA effector sequence homologous to host “reader” sequence will be investigated to determine the mechanism through which it exerts its function. This work will provide important insight into LANA’s fundamental function of episome maintenance, and may lead to novel strategies to prevent or treat KSHV associated malignancies.

卡波西肉瘤（KS）相关疱疹病毒（KSHV或HHV 8）是KS的病原体，原发性 渗出性淋巴瘤（PEL）和多中心Castleman病。这些肿瘤发生在艾滋病和其他 免疫力低下的状态。对于这些疾病没有特定的治疗方法。KS是领先的艾滋病 恶性肿瘤，在撒哈拉以南非洲流行。KS常累及口腔，可扩散至 内脏器官KSHV潜伏性感染肿瘤细胞，病毒基因组以环状、多拷贝形式存在， 染色体外，附加体（质粒）。为了在增殖细胞中持续存在，附加体必须首先复制 然后有效地分离成子核。肿瘤细胞活力依赖于潜伏的KSHV感染。 潜伏相关核抗原（拉娜）介导KSHV附加体的持续存在和这种功能 对于病毒的生存至关重要。因此，拉娜功能具有根本的重要性。拉娜代表 KSHV的致命弱点，因为它的破坏是致命的病毒。拉娜是KSHV的必要和充分条件 在没有其他病毒基因的情况下维持附加体。 Episome persistence由两个组成部分组成：1）病毒DNA的复制和2）病毒DNA的分离。 复制的附加体到后代细胞核中。拉娜介导这两种功能。C-末端拉娜结合 KSHV末端重复（TR）DNA中介导DNA复制的特异性序列。拉娜将TR DNA连接到 有丝分裂染色体分离基因组的女儿核。为了形成一个分子链， 结合结构域（DBD）结合TR DNA，N-LANA同时结合组蛋白H2 A/H2 B， 核小体的一部分。除了N-和C-拉娜外，内部拉娜序列对附加体也很重要 坚持不懈 我们发现了两个不同的内部拉娜区域，一个由独特的序列组成，另一个由不同的序列组成。 在重复序列中，这对拉娜介导的附加体持久性至关重要。的唯一序列 包含三个不同的功能基序，而重复区域与宿主细胞“阅读器”同源， 顺序这两个区域将在这项工作中进行调查。 这项工作将使用详细、深入的方法来调查最近确定的每一个拉娜 功能序列实验将使用一个严格开发的检测小组，以确定功能 基序和确定的机制，通过该独特的序列介导附加体持久性。的 将研究与宿主“阅读器”序列同源的重复拉娜效应子序列，以确定 通过它发挥其功能的机制。这项工作将提供重要的洞察拉娜的 附加体维持的基本功能，并可能导致预防或治疗KSHV的新策略 相关恶性肿瘤。