Investigating host protein interactions of the Human Papillomavirus E2 protein

研究人乳头瘤病毒 E2 蛋白的宿主蛋白相互作用

• 批准号: 8901724
• 负责人: Peris Nicole Bentley
• 金额: $ 3.52万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8901724
• 关键词: Antiviral Agents; Binding; Bovine Papillomavirus; Bovine papillomavirus E2 protein; Cell Culture Techniques; Cell Line; Cell Nucleus; Centromere; Chromatin; Chromosome Fragile Sites; Chromosomes; Collaborations; Complex; Computer software; DNA; DNA Damage; DNA Double Strand Break; DNA amplification; Data; Dimerization; Double Strand Break Repair; Event; Genome; HPV-High Risk; Human Papillomavirus; Human papilloma virus infection; Human papillomavirus 11; Human papillomavirus 16; Human papillomavirus 16 E1 protein; Individual; Infection; Life Cycle Stages; Low risk HPV; Maintenance; Mammalian Cell; Maps; Mass Spectrum Analysis; Mediating; Mitotic; Oncogenic; Papillomavirus; Papillomavirus Protein E2; Pathology; Pattern; Phase; Phenotype; Play; Prevention; Process; Proteins; Proteomics; Publishing; Recombinant DNA; Recruitment Activity; Research; Role; Sister Chromatid; Staging; Surveys; Techniques; Vaccines; Viral; Viral Genome; Virus; Western Blotting; comparative; ds-DNA; insight; medical schools; member; novel; oncogene protein E2, Human papillomavirus type 8; protein E2, Human papilloma virus type 1; protein complex; public health relevance; research study; response; tissue culture; viral DNA; virus host interaction

DESCRIPTION (provided by applicant): Preliminary data from Bovine Papillomavirus (BPV) MS/ComPASS experiments performed by Alvin Tan, a previous member of the Howley lab, have identified an uncharacterized interaction of BPV E2 with SMC5 and SMC6 (unpublished). The SMC5/6 complex is a part of the DNA damage response and is required for DNA double strand break repair and homologous recombination.41,42,43,45,47,48 The viral E1 protein induces a DNA damage response (DDR) in the nucleus and, together with E2, causes double strand breaks.18,19,20,56 E1- E2 replication foci contain DDR proteins and markers of homologous replication.19,56 I aim to determine whether interaction of SMC5/6 with E2 plays a role in viral replication or the localization of replication foci to vulnerable points in DNA, whic would be more susceptible to the integration of viral DNA. I have confirmed the interaction of HPV-16 and HPV-8 E2 with SMC6 through mammalian cell culture techniques and western blotting (unpublished). Papillomavirus E2 proteins of the Beta and Gamma genera, which includes HPV-8, display a unique binding pattern on host mitotic chromatin and unlike that of other genera, the host binding partner to chromatin is unknown.14,35,39 HPV-8 E2 and SMC5/6 share the same unique chromosomal association (to centromeres and rDNA loci), and thus I aim to determine whether the SMC5/6 complex is the principal tethering factor for these E2 proteins.48,50,54,65 Establishment of this complex in a tethering role during the genome maintenance phase, supports the hypothesis that it could mediate the concentration of viral genomes to fragile sites in host DNA upon induction of the host DDR, which occurs during the genome amplification phase. I also aim to use a proteomics approach to discover novel host-protein interactions of the HPV E2 protein and identify differences between the virus-host protein interactions of oncogenic and low-risk HPVs. I also aim to study the consequences of newly discovered interactions on the functions of E2 and the lifecycle and pathology of HPV. I plan to conduct a proteomic screen on a diverse group of E2 proteins. This technique will be comprised of mass spectrometry (MS) on E2 associated protein complexes, which will be co-immunoprecipitated from cell lines stably expressing these proteins, and subsequent Comparative Proteomic Analysis Software Suite (ComPASS) analysis in collaboration with the Harper lab at Harvard Medical School. This technique is expected to identify novel E2 high confidence interacting proteins unique to a particular HPV genus or species, or conserved across all HPV types. These techniques have been used successfully in previous studies in the Howley lab to uncover novel protein interactions of the HPV proteins E6 and E7.62,63,64 Through this proteomics technique, I aim to conduct a comprehensive survey of E2 binding partners.

描述（由申请人提供）：来自Howley Lab的先前成员Alvin Tan进行的牛乳头瘤病毒（BPV）MS/Compass实验的初步数据，已经确定了BPV E2与SMC5和SMC6和SMC6的未表征相互作用（未公开）。 SMC5/6复合物是DNA损伤响应的一部分，是DNA双链断裂修复和同源重组所必需的。41,42,43,45,45,47,48病毒E1蛋白在核中诱导DNA损伤响应（DDR），并与E2中的E2诱导E2，并将其与E2造成双链，并在E2中造成双重损失。同源复制的标记。19,56i的目的是确定SMC5/6与E2的相互作用是否在病毒复制中起作用，还是将复制焦点定位到DNA中脆弱点的位置，其对病毒DNA的整合更容易受到影响。 我已经通过哺乳动物细胞培养技术和蛋白质印迹（未发表）证实了HPV-16和HPV-8 E2与SMC6的相互作用。 Papillomavirus E2 proteins of the Beta and Gamma genera, which includes HPV-8, display a unique binding pattern on host mitotic chromatin and unlike that of other genera, the host binding partner to chromatin is unknown.14,35,39 HPV-8 E2 and SMC5/6 share the same unique chromosomal association (to centromeres and rDNA loci), and thus I aim to determine SMC5/6复合物是否是这些E2蛋白的主要束缚因子。48,50,54,65在基因组维持阶段的束缚作用中建立了该复合物，它支持以下假设：它可以介导宿主DDR诱导宿主DDR的基因组诱导的宿主DNA中的病毒基因组的浓度，该假设在基因组中的诱导阶段，该阶段是友好的阶段。 我还旨在使用蛋白质组学方法来发现HPV E2蛋白的新型宿主 - 蛋白质相互作用，并确定致癌和低风险HPV的病毒宿主蛋白相互作用之间的差异。我还旨在研究新发现的相互作用对E2功能以及HPV生命周期和病理的后果。我计划在各种E2蛋白质组上进行蛋白质组学筛选。该技术将由E2相关蛋白质复合物上的质谱（MS）组成，该技术将从稳定表达这些蛋白质的细胞系中共免疫沉淀，以及随后与哈佛医学院Harper Lab合作的比较蛋白质组学分析套件（Compass）软件套件（Compass）。预计该技术将确定具有特定HPV属或物种独有的新型E2高置信度相互作用，或在所有HPV类型中保守。这些技术已在霍利实验室的先前研究中成功使用，以通过这种蛋白质组学技术发现HPV蛋白E6和E7.62,63,64的新型蛋白质相互作用，我旨在对E2结合伙伴进行全面的调查。