Investigating host protein interactions of the Human Papillomavirus E2 protein

描述（由申请人提供）：由Howley实验室的前成员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的未表征的相互作用（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18,19,20,56E1- E2复制病灶含有DDR蛋白和同源复制标记物19,56我的目的是确定SMC5/6与E2的相互作用是否在病毒复制中发挥作用，或者将复制焦点定位到DNA中的易感点，后者更容易受到病毒DNA的整合。我已经通过哺乳动物细胞培养技术和western blotting（未发表）证实了HPV-16和hpv - 8e2与SMC6的相互作用。包括HPV-8在内的β和γ属乳头瘤病毒E2蛋白在宿主有丝分裂染色质上显示出独特的结合模式，与其他属不同的是，宿主与染色质的结合伙伴是未知的14,35,39HPV-8 E2和SMC5/6具有相同的独特染色体关联（到着丝粒和rDNA位点），因此我的目的是确定SMC5/6复合物是否是这些E2蛋白的主要束缚因子。48,50,54,65该复合体在基因组维持阶段具有拴索作用，这支持了一种假设，即在基因组扩增阶段发生宿主DDR诱导时，它可以介导病毒基因组集中到宿主DNA的脆弱位点。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相互作用，并确定致癌和低风险HPV病毒与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功能和HPV生命周期和病理的影响。我计划对不同组的E2蛋白进行蛋白质组学筛选。该技术将包括E2相关蛋白复合物的质谱分析（MS），这些蛋白将从稳定表达这些蛋白的细胞系中共同免疫沉淀，随后与哈佛医学院哈珀实验室合作进行比较蛋白质组学分析软件套件（ComPASS）分析。该技术有望鉴定出特定HPV属或物种特有的新型E2高置信度相互作用蛋白，或在所有HPV类型中保守。这些技术已经在Howley实验室的先前研究中成功地用于揭示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。

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Tan, a previous member of the Howley lab, have identified an uncharacterized interaction of BPV E2 with SMC5 and SMC6 (unpublished).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the interaction of HPV-16 and HPV-8 E2 with SMC6 through mammalian cell culture techniques and western blotting (unpublished).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.