Mechanisms of HPV DNA Segregation

HPV DNA 分离的机制

• 批准号: 7032920
• 负责人: LOUISE T CHOW
• 金额: $ 33.84万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 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/7032920
• 关键词: DNA replication; DNA replication origin; SDS polyacrylamide gel electrophoresis; cell cycle; chromatin immunoprecipitation; chromosome movement; confocal scanning microscopy; host organism interaction; human papillomavirus; mass spectrometry; matrix assisted laser desorption ionization; microtubule associated protein; plasmids; protein localization; protein protein interaction; protein transport; time resolved data; virus DNA; virus genetics; virus protein

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（由申请方提供）：人乳头瘤病毒（HPV）是医学上重要的病原体，通常可诱导上皮细胞良性过度增殖。然而，性传播的高危病毒基因型可导致宫颈和阴茎发育不良和癌症。HPV在循环基底和副基底角质形成细胞中建立持续的、通常是亚临床的感染，其中双链环状DNA基因组作为染色体外质粒以低拷贝数复制。在鳞状分化时发生生产性扩增。免疫抑制或手术/伤口愈合可以重新激活潜伏感染。没有有效的药物治疗。病毒DNA复制和持久性机制的表征对于开发抗病毒治疗策略至关重要，也因为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。我们开发了瞬时转染和无细胞系统来研究HPV DNA合成，这需要病毒起源（Ori）序列，Ori识别蛋白E2，DNA解旋酶E1和细胞DNA复制机制，以及伴侣蛋白和细胞周期蛋白E/cdk 2，一种对S期进入至关重要的激酶。为了确定当宿主细胞分裂时HPV DNA如何分离，我们构建了具有复制能力的GFP标记的HPV-11 E1和E2蛋白，并发现GFP-11 E2蛋白与有丝分裂纺锤体相关。六个具体目标是：（1）开发并验证一组融合到E1和E2以及融合到Gal 4 DNA结合结构域的活彩色荧光蛋白（FP），其能够追踪含有40个拷贝的Gal 4结合位点的修饰的Ori质粒。我们建议，以验证Ori DNA分离协会与有丝分裂纺锤体介导的E2。(2)表征HPV中纺锤体相关Ori DNA分离所需的任何其他序列元件。(3)使用质谱法鉴定介导E2-微管相互作用的细胞蛋白质。(4)通过体内共定位和体外相互作用证实E2与细胞内蛋白质的相互作用。(5)确定E2中负责这些相互作用的肽基序，并检查E2中的定点突变与有丝分裂纺锤体相关并介导DNA分离的能力。(6)确定FP标记的E1和E2和Ori DNA在非复制和复制条件下相对于已知核结构域如ND 10（PML）体和Cajal体的亚细胞地址。它们的运输将被捕获在活细胞作为延时电影使用多光子激光共聚焦扫描显微镜。这些研究将提供HPV DNA在体内复制的详细分子画像。预期的结果是一个明确的定义染色体外质粒分离和维护的机制。