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Tan, a previous member of the Howley lab, have identified an uncharacterized interaction of BPV E2 with SMC5 and SMC6 (unpublished).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)：由Howley实验室前成员Alvin Tan进行的牛乳头状瘤病毒(BPV)MS/COMPASS实验的初步数据发现，BPVE2与SMC5和SMC6存在未知的相互作用(未发表)。SMC5/6复合体是DNA损伤反应的一部分，是DNA双链断裂修复和同源重组所必需的。41，42，43，45，47，48病毒的E1蛋白在细胞核中诱导DNA损伤反应(DDR)，并与E2一起引起双链断裂。18，19，20，56个E1-E2复制点含有DDR蛋白和同源复制的标志。19，56I旨在确定SMC5/6与E2的相互作用是否在病毒复制中发挥作用，或者将复制点定位到DNA中更容易整合的易受病毒DNA整合的复制点。我已经通过哺乳动物细胞培养技术和Western blotting证实了HPV-16和HPV-8 E2与SMC6的相互作用(未发表)。包括HPV-8在内的Beta和Gamma属的乳头瘤病毒E2蛋白在宿主有丝分裂染色质上表现出独特的结合模式，与其他属不同的是，宿主与染色质的结合伙伴尚不清楚。14，35，39 HPV-8 E2和SMC5/6具有相同的独特染色体联系(与着丝粒和rDNA位点)，因此我旨在确定SMC5/6复合体是否是这些E2蛋白的主要系留因子。48，50，54，65在基因组维持阶段，该复合体的建立起到了系留作用，支持了这样的假设，即它可以在宿主DDR，诱导宿主DNA中的脆性位置时调节病毒基因组的浓度这发生在基因组扩增阶段。我还打算使用蛋白质组学的方法来发现HPVE2蛋白的新的宿主-蛋白质相互作用，并识别致癌和低风险HPV的病毒-宿主蛋白相互作用之间的差异。我还打算研究新发现的相互作用对E2功能以及HPV的生命周期和病理的影响。我计划对一组不同的E2蛋白进行蛋白质组筛选。这项技术将包括对E2相关蛋白质复合体的质谱仪(MS)，它将从稳定表达这些蛋白质的细胞系中免疫共沉淀，以及随后与哈佛医学院哈珀实验室合作的比较蛋白质分析软件套件(COMPASS)分析。这项技术有望识别新的E2高置信度相互作用蛋白，这些蛋白是特定HPV属或物种独有的，或在所有HPV类型中保守的。这些技术已经在Howley实验室以前的研究中成功地用于发现HPV蛋白E6和E7.62，63，64的新的蛋白质相互作用。通过这种蛋白质组学技术，我的目标是对E2结合伙伴进行全面的调查。