Mechanisms of HPV DNA Segregation

描述(申请人提供)：人乳头瘤病毒(HPV)是医学上重要的病原体，通常会导致良性的上皮细胞过度增殖。然而，性传播的高危病毒基因型会导致宫颈和阴茎发育不良以及癌症。HPV在循环的基底和基底旁角质形成细胞中建立持续的、通常是亚临床的感染，在那里双链、环状DNA基因组以低拷贝数作为染色体外质粒复制。生产性扩增发生在鳞状细胞分化之后。免疫抑制或手术/伤口愈合可以重新激活潜伏感染。目前还没有有效的药物治疗方法。病毒DNA复制和持续机制的表征对于抗病毒治疗策略的发展至关重要，也因为HPV为真核细胞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。我们建立了瞬时转染和无细胞系统来研究HPVDNA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，这是进入S时相的关键激酶。为了确定当宿主细胞分裂时HPV DNA是如何分离的，我们构建了具有复制能力的GFP标记的HPV-11E1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)开发和验证一组与E1和E2以及Gal4 DNA结合域融合的活的彩色荧光蛋白(FP)，这使得能够追踪含有40个Gal4结合位点的修饰的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相关联来分离。(2)鉴定纺锤体相关的OrI DNA分离所需的HPV中的其他序列元件。(3)用质谱仪鉴定参与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与细胞蛋白的相互作用。(5)确定E2中负责这些相互作用的多肽基序，并检测E2中的定点突变，以了解它们与有丝分裂纺锤体结合和介导DNA分离的能力。(6)确定在非复制和复制条件下，相对于ND10(PML)小体和Cajal小体等已知核结构域，FP标记的E1和E2以及OrI DNA的亚细胞地址。它们的贩运将通过使用多光子激光共聚焦扫描显微镜的延时电影在活细胞中被捕捉到。这些研究将提供HPV DNA在体内复制的详细分子画像。预期的结果是对染色体外质粒分离和维持的机制有一个明确的定义。

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.