Mechanisms of HPV DNA Segregation

HPV DNA 分离的机制

基本信息

批准号：
7224164
负责人：
LOUISE T CHOW
金额：
$ 33.85万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-04-01 至 2009-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7224164
关键词：
Address Affinity Chromatography Alanine Antibodies Antiviral Therapy Anus Benign Binding Binding Sites Biological Assay Biological Models Carcinoma Cell Cycle Cell Line Cell-Free System Cells Centrosome Cervical Chimeric Proteins Circular DNA Color Complex Condition Confocal Microscopy Cyclin E DNA DNA Binding Domain DNA Markers DNA Tumor Viruses DNA biosynthesis DNA chemical synthesis DNA-Directed DNA Polymerase Development Dysplasia Epithelium Eukaryota Eukaryotic Cell Event Family Fluorescence Microscopy Genetic Genetic Transcription Genome Genotype Green Fluorescent Proteins Human Human Papillomavirus Image Immunosuppression In Vitro Indium Individual Infection Interphase Cell Investigation Laser Scanning Confocal Microscopy Lasers Lead Life Localized MALDI-TOF Mass Spectrometry Maintenance Malignant Neoplasms Malignant neoplasm of pharynx Mammalian Cell Maps Mass Spectrum Analysis Mediating Metaphase Microtubule-Associated Proteins Microtubules Mitosis Mitotic Mitotic Spindle Apparatus Mitotic spindle Molecular Molecular Chaperones Movement Mutate Mutation Nuclear Nucleic Acid Regulatory Sequences Numbers Oncogenic Operative Surgical Procedures Optics Outcome PCNA gene Papillomavirus Peptides Phase Phosphotransferases Plasmids Portraits Protein Binding Proteins Relative (related person)Replicon Risk Scanning Series Site Squamous Differentiation Squamous Epithelium Surrogate Markers Tertiary Protein Structure Testing Therapeutic immunosuppression Time Topoisomerase Transfection Translations Tubulin Viral Viral Proteins Wound Healing Yeasts chromatin immunoprecipitation chromosome replication digital imaging enhanced green fluorescent protein genetic regulatory protein helicase in vivo keratinocyte latent infection movie multi-photon novel pathogen plasmid DNA polyacrylamide gels reconstitution segregation time use trafficking tumor progression viral DNA

项目摘要

DESCRIPTION (provided by applicant): Human papillomaviruses (HPVs) are medically important pathogens that normally induce benign hyperproliferation of epithelia. However, the sexually transmitted high-risk viral genotypes can cause cervical and penile dysplasias and cancer. HPVs establish persistent, often subclinical, infections in cycling basal and parabasal keratinocytes, where the double-stranded, circular DNA genome replicates as extra-chromosomal plasmids at low copy number. Productive amplification takes place upon squamous differentiation. Immunosuppression or surgery/wound healing can reactivate latent infections. There is no effective pharmacologic treatment. Characterization of the mechanisms of viral DNA replication and persistence is of paramount importance to the development of strategies for antiviral therapies and also because HPV provides a simple but elegant model system for replication and chromosome dynamics in eukaryotes. We developed both transient transfection and cell-free systems to study HPV DNA synthesis, which requires the viral origin (Ori) sequences, the Ori recognition protein E2, the DNA helicase E1 and the cellular DNA replication machinery, as well as chaperones and cyclin E/cdk2, a kinase critical for S phase entry. To determine how HPV DNA segregates when the host cells divide, we constructed replication-competent GFP-tagged HPV-11 E1 and E2 proteins and discovered that GFP-11E2 protein associates with mitotic spindles. Six Specific Aims are: (1) To develop and validate a set of living color fluorescent proteins (FP) fused to E1 and E2, as well as to the Gal4 DNA binding domain, which enables tracing of a modified Ori plasmid containing 40 copies of the Gal4 binding site. We propose to verify that Ori DNA segregates by association with mitotic spindles mediated by E2. (2) To characterize any other sequence elements in HPV necessary for spindle-associated Ori DNA segregation. (3) To identify cellular proteins that mediate the E2-microtubule interaction, using mass spectrometry. (4) To confirm the interactions between E2 and cellular proteins by in vivo colocalization and in vitro interaction. (5) To identify peptide motifs in E2 responsible for these interactions and to examine site-directed mutations in E2 for their abilities to associate with the mitotic spindles and to mediate DNA segregation. (6) To identify the subcellular addresses of FP-tagged E1 and E2 and Ori DNA under nonreplicating and replicating conditions relative to known nuclear domains such as ND10 (PML) bodies and Cajal bodies. Their trafficking will be captured in live cells as time-lapse movies using multi-photon laser confocal scanning microscopy. These investigations will provide a detailed molecular portrait of HPV DNA replication in vivo. The anticipated outcome is a clear definition of the mechanisms of extrachromosomal plasmid segregation and maintenance.

描述（由申请人提供）：人乳头瘤病毒（HPV）是通常诱导上皮良性过度增殖的医学重要病原体。但是，性传播的高风险病毒基因型会引起宫颈和阴茎发育不良和癌症。 HPV在循环基底和副副膜角质形成细胞中建立持续的，通常是亚临床感染，其中双链的圆形DNA基因组在低拷贝数中以外染色体质粒的形式复制。鳞状分化时会发生生产性扩增。免疫抑制或手术/伤口愈合可以重新激活潜在感染。没有有效的药理治疗。病毒DNA复制和持久性机制的表征对于制定抗病毒疗法的策略至关重要，也是因为HPV为真核生物中的复制和染色体动力学提供了简单但优雅的模型系统。我们开发了瞬态转染和无细胞的系统来研究HPV DNA合成，它需要病毒起源（ORI）序列，ORI识别蛋白E2，DNA解旋酶E1和细胞DNA复制机制，以及伴侣E/CDK2，伴侣E/CDK2，对S相位的依赖的Kinase。为了确定HPV DNA在宿主细胞分裂时如何分离，我们构建了具有复制功能的GFP标记的HPV-11 E1和E2蛋白，并发现GFP-11E2蛋白与有丝分裂纺锤体相关。六个具体目的是：（1）开发和验证一组融合到E1和E2的生活色彩荧光蛋白（FP），以及GAL4 DNA结合结构域，该结构域可以追踪一个经过修改的ORI质粒，该质粒包含40个GAL4结合位点的副本。我们建议通过与E2介导的有丝分裂纺锤体相关性来验证ORI DNA分离。（2）表征与纺锤体相关的ORI DNA分离所需的HPV中的任何其他序列元素。（3）鉴定使用质谱法介导E2微管相互作用的细胞蛋白。（4）通过体内共定位和体外相互作用来确认E2与细胞蛋白之间的相互作用。（5）确定负责这些相互作用的E2中的肽基序，并检查E2中的位置导向突变，以使其与有丝分裂纺锤体结合并介导DNA分离的能力。（6）在非复制和复制条件下，相对于已知的核域（如ND10（PML）身体和Cajal体），在非重复和复制条件下确定了FP标签的E1和E2和ORI DNA的亚细胞地址。使用多光子激光共聚焦扫描显微镜作为延时电影，将在现场细胞中捕获他们的贩运。这些研究将提供体内HPV DNA复制的详细分子肖像。预期的结果是对外质体质粒分离和维持机制的明确定义。