Mechanisms of Human Papillomavirus DNA Replication

描述(由申请人提供)：人乳头瘤病毒(HPV)组成了一个具有重要医学意义的人类病原体大家族。这些小的DNA病毒建立了鳞状上皮的持续性感染，通常会导致良性的增生性病变。高危基因感染可进展为不典型增生和癌症，特别是宫颈癌和阴茎癌。在循环的基底角质形成细胞中，双链环状基因组以染色体外质粒的形式以低拷贝数复制。只有在分化棘状细胞时才能进行生产性扩增。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复合体，并且E1的活性受多个激酶调节。我们的初步结果显示，一些已知的控制染色体DNA复制的细胞蛋白也调节无细胞复制系统中的HPV复制，包括CDT1、Gminin、TopBP1和P53。双黄素抑制CDT1，CDT1是将细胞复制解旋酶MCM复合体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的复制。TopBP1参与细胞DNA复制和修复。它与DNA聚合酶epsilon相互作用，epsilon是一种具有关键但尚未阐明的功能的聚合酶。TopBP1已被报道与HPV16E2相互作用，并刺激HPV瞬时复制。我们现在证明TopBP1增强了HPV的无细胞复制。P53是HPV E6蛋白失活的靶标，已知可以与E2结合并抑制病毒的瞬时扩增复制。我们证明，在存在或不存在E2的情况下，P53抑制HPV复制，这表明了其他机制。我们认为，在每一种情况下，无细胞系统都提供了一个很好的机会来阐明病毒和细胞DNA复制的调节机制。我们提出了四个具体目标。1.探索HPV DNA复制与调控的新模型。该模型可以解释病毒DNA在感染组织中复制的维持模式和扩增模式。2.探讨TopBP1和DNA聚合酶epsilon在HPV DNA复制中的作用。3.为了验证我们的假设，即P53抑制HPV DNA复制至少部分是通过干扰重组蛋白的活性来实现的，这些重组蛋白被认为与染色体DNA复制有关。4.为了阐明E1的磷酸化如何调节其复制功能，是本研究的继续。

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.