Genetic and Biochemical Studies of KSHV LANA

KSHV LANA 的遗传和生化研究

• 批准号: 10025546
• 负责人: Kenneth M Kaye
• 金额: $ 70.7万
• 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/10025546
• 关键词: Acquired Immunodeficiency Syndrome; Africa South of the Sahara; Animal Model; Binding; Biochemical; Biochemical Genetics; Biological; Biological Assay; Blood group antigen S; C-terminal; Cell Nucleus; Cell Survival; Cells; DNA; DNA Binding Domain; DNA biosynthesis; Daughter; Development; Disease; Elements; Epidemic; Episome; Etiology; Genes; Genetic; Genome; Glycogen Synthase Kinase 3; Herpesviridae; Herpesviridae Infections; Histone H2A; Human; Human Herpesvirus 8; Immunocompromised Host; Investigation; Kaposi Sarcoma; Lead; 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.

Kaposi肉瘤(KS)相关疱疹病毒(KSHV或HHV8)是原发KS的病原体 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疾病中 免疫受损状态。目前还没有针对这些疾病的特效药。KS是艾滋病的头号杀手 恶性，并在撒哈拉以南非洲流行。KS通常累及口腔，并可传播到 内脏器官。KSHV潜伏感染肿瘤细胞，病毒基因组以环状、多拷贝、 染色体外，上染色体(质粒)。为了维持细胞的增殖，上皮质体必须首先进行复制 然后有效地分离到子核。肿瘤细胞的存活依赖于潜伏的KSHV感染。 潜伏期相关核抗原(LANA)介导KSHV流行体持续及其作用 对病毒的生存至关重要。因此，LANA功能是至关重要的。拉娜代表着 KSHV的致命弱点是它的破坏对病毒是致命的。Lana是KSHV的充要条件 在没有其他病毒基因的情况下保持流行状态。 Episome持久性由两个部分组成：1)病毒DNA的复制和2)分离 复制的上体到子代细胞核。拉娜调停了这两种功能。C-终端LANA绑定 KSHV末端重复序列(TR)DNA中的特定序列介导DNA复制。拉娜将TR DNA捆绑到 有丝分裂染色体将基因组分离到子核。为了形成分子纽带，C-LANA DNA 结合结构域(DBD)可与TRDNA结合，N-LANA可同时在折叠区域结合组蛋白H_2A/H_2B 核小体的部分。除了N-LANA和C-LANA，内部LANA序列对Episome也是至关重要的 坚持不懈。 我们发现了两个不同的内部LANA区域，一个由唯一序列组成，另一个 在重复序列中，这对LANA介导的Episome持久性至关重要。独特的序列 包含三个不同的功能基序，而重复区域与宿主细胞的“读取器”同源。 序列。这两个地区都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发现的每一个LANA 功能序列。实验将使用严格开发的化验小组来确定功能 基序，并确定独特的序列介导Episome持久性的机制。这个 将研究与宿主“读取器”序列同源的重复LANA效应序列，以确定 它发挥作用的机制。这项工作将为LANA提供重要的见解 Episome维护的基本功能，并可能导致预防或治疗KSHV的新策略 相关的恶性肿瘤。