Mechanisms of HPV DNA Segregation

描述（由申请人提供）：人乳头瘤病毒（HPV）是医学上重要的病原体，通常会诱导上皮细胞的良性过度增殖。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 基因组以低拷贝数复制为染色体外质粒。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抗病毒治疗策略的制定至关重要，而且还因为 HPV 为真核生物中的复制和染色体动力学提供了一个简单而优雅的模型系统。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，以及伴侣和细胞周期蛋白 E/cdk2（一种对进入 S 期至关重要的激酶）。为了确定宿主细胞分裂时 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-11E2 蛋白与有丝分裂纺锤体相关。六个具体目标是：(1) 开发并验证一组与 E1 和 E2 以及 Gal4 DNA 结合域融合的活彩色荧光蛋白 (FP)，从而能够追踪包含 40 个 Gal4 结合位点拷贝的修饰 Ori 质粒。我们建议验证 Ori DNA 通过与 E2 介导的有丝分裂纺锤体关联进行分离。 (2) 表征 HPV 中与纺锤体相关的 Ori DNA 分离所必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。 (4)通过体内共定位和体外相互作用证实E2与细胞蛋白之间的相互作用。 (5) 鉴定 E2 中负责这些相互作用的肽基序，并检查 E2 中的定点突变，以确定它们与有丝分裂纺锤体关联并介导 DNA 分离的能力。 (6) 识别 FP 标记的 E1 和 E2 以及 Ori DNA 在非复制和复制条件下相对于已知核结构域（例如 ND10 (PML) 小体和 Cajal 小体）的亚细胞地址。它们的贩运将使用多光子激光共焦扫描显微镜在活细胞中以延时电影的形式捕获。这些研究将提供 HPV DNA 在体内复制的详细分子肖像。预期结果是对染色体外质粒分离和维持机制的明确定义。

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.