Molecular basis and regulation of the DNA binding activity of KSHV LANA

KSHV LANA DNA结合活性的分子基础和调控

• 批准号: 8394018
• 负责人: John F Domsic
• 金额: $ 5.22万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8394018
• 关键词: Affect; Affinity; Amino Acids; B-Lymphocytes; Base Pairing; Binding; Binding Sites; Biological Assay; C-terminal; Cell Culture Techniques; Cell division; Cells; Chromosomes; Complement; Complex; Crystallization; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; DNA biosynthesis; Data; Data Set; Development; Disease; Electrophoretic Mobility Shift Assay; Ensure; Episome; Epstein-Barr Virus Nuclear Antigens; Fluorescence Polarization; Foundations; Future; Gene Expression; Genes; Genetic Transcription; Genome; Goals; Heavy Metals; Herpesviridae; Herpesviridae Infections; Housing; Human; Human Herpesvirus 8; Immune response; In Vitro; Kaposi Sarcoma; Laboratories; Life Cycle Stages; Lymphatic Endothelial Cells; Lymphoma; Lytic; Maintenance; Malignant - descriptor; Mammalian Cell; Mass Spectrum Analysis; Mediating; Methionine; Methods; Modification; Molecular; Multicentric Angiofollicular Lymphoid Hyperplasia; Mutation; N-terminal; Nucleosomes; Peptides; Phase; Phosphorylation; Plasmids; Play; Positioning Attribute; Post-Translational Protein Processing; Production; Protein Region; Proteins; Protocols documentation; RNA Splicing; Regulation; Relative (related person); Research; Resolution; Role; Series; Serine; Serine Phosphorylation Site; Specificity; Structure; Techniques; Terminal Repeat Sequences; Testing; Transcript; Viral; Viral Genes; Viral Genome; Virus Diseases; Work; X ray diffraction analysis; X-Ray Diffraction; antigen binding; base; daughter cell; design; effusion; human disease; improved; inhibitor/antagonist; insight; latency-associated nuclear antigen; latent infection; latent persistent infection; mimetics; mutant; novel; pathogen; prevent; promoter; recombinant virus; small molecule; three dimensional structure

DESCRIPTION (provided by applicant): Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is the etiological agent of several human diseases, including Kaposi's sarcoma, primary effusion lymphoma, and a subset of multicentric Castleman's disease. KSHV infections exist in two states: a non-productive latent state and a proliferative lytic state. During latency, viral gene expression focuses on those genes that encode products required for preventing a host immune response and maintaining a state of latency. To ensure that viral infection persists after cell division, the viral genome is circularized as an episome and tethered to the host chromosome by the protein latency-associated nuclear antigen (LANA). LANA is a multi-functional protein that plays a pivotal role in maintaining passage of the episome as well as repressing lytic reactivation and host immune response. The N-terminus of LANA interacts with a host nucleosome while the C-terminus binds sequence specifically to a portion of the viral episome. The structure of an N-terminal peptide bound to the nucleosome has previously been solved; however, no structural information is available on the C-terminus. To understand how LANA mediates this sequence specific interaction we propose to determine the three-dimensional crystal structure of the LANA DNA binding domain both alone and bound to its DNA binding site. Utilizing this structural information we will then create mutations in regions that we identify to e important for sequence specificity and DNA binding function. Also, we have identified novel serine phosphorylation sites within the C-terminus that have been suggested to play a role in interactions with cellular proteins and may have an effect on DNA binding. We propose to make mutations of these serines to either abrogate or mimic a constitutive state of phosphorylation. We will utilize previously developed assays to test these mutations for DNA binding, episome maintenance, and genome replication. The information obtained from these studies will provide a foundation for the development of small-molecule inhibitors of LANA as well as an understanding of the role of post-translational modifications in DNA binding and the viral life cycle. PUBLIC HEALTH RELEVANCE: Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is a human viral pathogen that is the causative agent of several lymphomas. The KSHV genome is persistent in infected cells and is directly attached to the host chromosome. The goal of this project is to determine the three-dimensional structure of the viral genome binding portion of the protein LANA, which is responsible for attaching the viral genome to the host chromosome and to understand how modifications to this protein affect its DNA binding function.

描述（由申请方提供）：卡波西肉瘤相关疱疹病毒（KSHV）是几种人类疾病的病原体，包括卡波西肉瘤、原发性渗出性淋巴瘤和多中心Castleman病的一个子集。KSHV感染存在两种状态：非生产性潜伏状态和增殖性溶解状态。在潜伏期，病毒基因表达集中在那些编码防止宿主免疫反应和维持潜伏状态所需产物的基因上。为了确保病毒感染在细胞分裂后持续存在，病毒基因组作为附加体被环化，并通过蛋白潜伏相关核抗原（拉娜）被拴系到宿主染色体。拉娜是一种多功能蛋白，在维持附加体的通过以及抑制裂解再活化和宿主免疫应答中起关键作用。拉娜的N-末端与宿主核小体相互作用，而C-末端序列特异性地结合病毒附加体的一部分。与核小体结合的N-末端肽的结构以前已经解决;然而，没有关于C-末端的结构信息。为了理解拉娜如何介导这种序列特异性相互作用，我们建议确定单独的和与其DNA结合位点结合的拉娜DNA结合结构域的三维晶体结构。利用这些结构信息，我们将在我们确定对序列特异性和DNA结合功能重要的区域中产生突变。此外，我们已经确定了新的丝氨酸磷酸化位点内的C-末端，已被建议发挥作用，与细胞蛋白质的相互作用，并可能对DNA结合的影响。我们建议使这些丝氨酸突变，以消除或模拟磷酸化的组成状态。我们将利用之前开发的检测方法来测试这些突变的DNA结合、附加体维持和基因组复制。从这些研究中获得的信息将为开发拉娜的小分子抑制剂以及理解DNA结合和病毒生命周期中的翻译后修饰的作用提供基础。 公共卫生相关性：卡波西肉瘤相关疱疹病毒（KSHV）是一种人类病毒病原体，是几种淋巴瘤的病原体。KSHV基因组在感染细胞中持续存在，并直接附着在宿主染色体上。该项目的目标是确定蛋白质拉娜的病毒基因组结合部分的三维结构，该蛋白质负责将病毒基因组附着到宿主染色体上，并了解对该蛋白质的修饰如何影响其DNA结合功能。