Control of Papillomavirus Expression and Transformation

描述（由申请人提供）：E2蛋白作为稳定的插曲体调节乳头瘤病毒基因组的转录和复制。它的n端结构域结合这些功能所必需的细胞因子和复合物。这些细胞蛋白通过E2 c末端结构域靶向病毒基因组，该结构域与病毒DNA中的特定序列具有高亲和力。这项拨款提案的目标是鉴定和表征细胞蛋白和E2激活其多种功能的途径。第一个目的是研究E2在细胞周期中的活性，当E2在S期将病毒E1 DNA解旋酶定位到原点时，以及在有丝分裂中，当E2将病毒基因组系在宿主染色体上，介导其分裂，并且正如我们在这里提出的那样，废除可能限制宿主细胞活力的有丝分裂检查点时。我们证明E2与几种有丝分裂蛋白的相互作用将在本研究中进一步表征。这些蛋白-蛋白相互作用揭示了病毒基因组在有丝分裂过程中附着于染色体的新机制。其他相互作用提示了在有丝分裂纺锤体上运输病毒基因组的机制。本研究的另一个目的是研究E2抑制有丝分裂检查点的能力。在一个例子中，我们测试了E2抑制细胞分裂的假设，细胞分裂可能是分化上皮细胞中病毒基因组扩增所必需的功能。病毒基因组整合后E2功能的丧失和E2的破坏可能是致癌性人乳头瘤病毒肿瘤进展的重要一步。目的2继续我们对转录调控机制的研究，特别强调E2如何克服体内染色质抑制。我们验证了E2与Swi/Snf染色质重塑复合体组分的相互作用是转录激活和抑制所必需的假设。这些研究将拓宽我们对E2在病毒复制程序中的分子功能的理解。

DESCRIPTION (provided by applicant): The E2 protein regulates transcription and replication of the papillomavirus genome as a stable episome. Its N-terminal domain binds cellular factors and complexes necessary for these functions. These cellular proteins are targeted to the viral genome by the E2 C-terminal domain, which binds with high affinity to specific sequences in the viral DNA. The goal of this grant proposal is to identify and characterize the cellular proteins and pathways through which E2 actuates its diverse functions. The first Aim investigates the activities of E2 during cell cycle, when E2 localizes the viral E1 DNA helicase to the origin in S phase, and in mitosis when E2 tethers the viral genome to host chromosomes, mediates its partitioning, and as we propose here, abrogates mitotic checkpoints that might otherwise restrict host cell viability. We demonstrate E2 interactions with several mitotic proteins that will be further characterized in this Aim. These protein- protein interactions reveal novel mechanisms for viral genome attachment to chromosomes during mitosis. Other interactions suggest a mechanism to transport the viral genome on the mitotic spindle. Another goal of this aim is to investigate the ability of E2 to inhibit mitotic checkpoints. In one example, we test the hypothesis that E2 inhibits cytokinesis, a function that is probably necessary for viral genome amplification in differentiated epithelial cells. Loss of these E2 functions following viral genome integration and disruption of E2 may represent an important step in neoplastic progression by oncogenic human papillomaviruses. Aim 2 continues our investigations of the mechanism of transcriptional regulation, with particular emphasis on how E2 overcomes chromatin repression in vivo. We test the hypothesis that E2 interactions with components of the Swi/Snf chromatin-remodeling complex are necessary for transcriptional activation and repression. These studies will broaden our understanding of the molecular functions of E2 in the viral replicative program. Lay Statement: Papillomaviruses cause benign infections that can become malignant, and are the etiologic agent of cervical cancer. The E2 protein controls essential functions of the virus. We propose to identify the proteins in the cell that interact with E2 and their functions. These are involved in fundamental process including gene expression and DNA replication. Understanding how E2 operates will provide insights into the biology of these pathways and infer how aberrations may occur that lead to the development of cancer.