Mechanisms of Human Papillomavirus DNA Replication

人乳头瘤病毒 DNA 复制机制

• 批准号: 6920580
• 负责人: LOUISE T CHOW
• 金额: $ 35万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6920580
• 关键词: DNA directed DNA polymerase; DNA replication; DNA replication origin; basal cell; cell free system; cell line; enzyme activity; helicase; host organism interaction; human papillomavirus; keratinocyte; molecular chaperones; p53 gene /protein; phosphorylation; protein protein interaction; protein purification; site directed mutagenesis; virus DNA; virus protein; virus replication

DESCRIPTION (provided by applicant): Human papillomaviruses (HPVs) comprise an extended family of medically important human pathogens. These small DNA viruses establish persistent infections of squamous epithelia, typically causing benign hyperproliferative lesions. Infections by higher risk genotypes can progress to dysplasias and carcinomas, notably cervical and penile cancers. The double-stranded, circular genome replicates as extrachromosomal plasmids at low copy number in cycling basal keratinocytes. Productive amplification takes place only in differentiating spinous cells. We and others developed transient and cell-free systems to study the mechanisms of HPV DNA replication and showed that replication requires an origin of replication (ori), the HPV ori recognition protein E2, the viral replicative helicase E1, the cellular DNA replication machinery and cyclin/cdk complexes and that the E1 activity is regulated by multiple kinases. Our preliminary results reveal that a number of cellular proteins known to control chromosomal DNA replication also regulate HPV replication in the cell-free replication system, including Cdt1, geminin, TopBP1, and p53. Geminin inhibits Cdt1, which is necessary for loading the cellular replicative helicase MCM complex onto the cellular chromatin. Our results implicate MCM in controlling HPV replication when E1 concentration is low. TopBP1 is involved in both cellular DNA replication and repair. It interacts with DNA polymerase epsilon, a polymerase with critical but yet-to-be elucidated functions. TopBP1 has been reported to interact with HPV16 E2 and to stimulate transient HPV replication. We now show that TopBP1 enhances HPV cell-free replication. p53 is targeted by HPV E6 protein for inactivation and is known to bind E2 and inhibit viral transient amplification replication. We demonstrate that p53 inhibits HPV replication in the presence or absence of E2, indicative of additional mechanisms. We suggest that, in each case, the cell-free system provides an excellent opportunity to elucidate the mechanisms of regulation for both viral and cellular DNA replication. We propose four specific aims. 1. To test a new model for HPV DNA replication and regulation. This model would account for the maintenance mode and the amplification mode of viral DNA replication in infected tissues. 2. To determine the roles of TopBP1 and DNA polymerase epsilon in HPV DNA replication. 3. To test our hypothesis that p53 inhibition of HPV DNA replication is mediated at least in part by interfering with the activities of recombination proteins that have been hypothesized to be involved in chromosomal DNA replication. 4. To elucidate how E1 phosphorylation modulates its replication functions, a continuation of present research.

描述（由申请人提供）：人乳头瘤病毒（HPV）包含一个医学上重要的人类病原体的大家族。这些小 DNA 病毒对鳞状上皮细胞造成持续感染，通常会导致良性过度增殖性病变。高风险基因型的感染可能发展为不典型增生和癌症，特别是宫颈癌和阴茎癌。双链环状基因组在循环基底角质形成细胞中以低拷贝数复制为染色体外质粒。生产性扩增仅发生在分化的棘细胞中。我们和其他人开发了瞬时和无细胞系统来研究 HPV DNA 复制的机制，并表明复制需要复制起点 (ori)、HPV ori 识别蛋白 E2、病毒复制解旋酶 E1、细胞 DNA 复制机制和细胞周期蛋白/cdk 复合物，并且 E1 活性受多种激酶调节。我们的初步结果表明，许多已知控制染色体 DNA 复制的细胞蛋白也在无细胞复制系统中调节 HPV 复制，包括 Cdt1、geminin、TopBP1 和 p53。 Geminin 抑制 Cdt1，这是将细胞复制解旋酶 MCM 复合物加载到细胞染色质上所必需的。我们的结果表明，当 E1 浓度较低时，MCM 可以控制 HPV 复制。 TopBP1 参与细胞 DNA 复制和修复。它与 DNA 聚合酶 epsilon 相互作用，这是一种具有关键但尚未阐明功能的聚合酶。据报道，TopBP1 与 HPV16 E2 相互作用并刺激短暂的 HPV 复制。我们现在证明 TopBP1 增强 HPV 无细胞复制。 p53 是 HPV E6 蛋白失活的靶标，已知它可以结合 E2 并抑制病毒瞬时扩增复制。我们证明 p53 在存在或不存在 E2 的情况下抑制 HPV 复制，表明存在其他机制。我们认为，在每种情况下，无细胞系统都为阐明病毒和细胞 DNA 复制的调节机制提供了绝佳的机会。我们提出四个具体目标。 1. 测试HPV DNA复制和调控的新模型。该模型将解释受感染组织中病毒 DNA 复制的维持模式和扩增模式。 2. 确定TopBP1和​​DNA聚合酶epsilon在HPV DNA复制中的作用。 3. 检验我们的假设，即 p53 对 HPV DNA 复制的抑制至少部分是通过干扰重组蛋白的活性来介导的，而重组蛋白被假设参与染色体 DNA 复制。 4. 阐明E1磷酸化如何调节其复制功能，这是当前研究的延续。