Investigating host protein interactions of the Human Papillomavirus E2 protein

描述（由申请方提供）：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SMC 5和SMC 6的未表征的相互作用（未发表）。SMC 5/6复合物是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E2一起导致双链断裂。18，19，20，5/6 E1- E2复制灶含有DDR蛋白和同源复制的标记。19，5/6 I旨在确定SMC 5/6与E2的相互作用是否在病毒复制中起作用或复制灶定位于DNA中的易损点，更容易被病毒DNA整合 我已经通过哺乳动物细胞培养技术和蛋白质印迹法证实了HPV-16和HPV-8 E2与SMC 6的相互作用（未发表）。β和γ属的乳头瘤病毒E2蛋白（包括HPV-8）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，宿主与染色质的结合伴侣是未知的。14，35，39 HPV-8 E2和SMC 5/6具有相同的独特染色体关联（着丝粒和rDNA位点），因此我的目的是确定SMC 5/6复合物是否是这些E2蛋白的主要束缚因子。48，50，54，65在基因组维持阶段期间以系留作用建立该复合物支持了以下假设：在基因组扩增阶段期间发生的宿主DDR诱导后，其可介导病毒基因组向宿主DNA中的脆性位点浓缩。 我还打算使用蛋白质组学方法来发现HPV E2蛋白的新宿主蛋白相互作用，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生命周期和病理学的影响。我计划对一组不同的E2蛋白进行蛋白质组学筛选。该技术将包括对E2相关蛋白复合物的质谱分析（MS），其将从稳定表达这些蛋白的细胞系中共免疫沉淀，以及随后与哈佛医学院的哈珀实验室合作进行的比较蛋白质组学分析软件套件（ComPASS）分析。该技术有望鉴定特定HPV属或种所特有的或在所有HPV类型中保守的新型E2高置信度相互作用蛋白。这些技术已成功地用于Howley实验室以前的研究中，以揭示HPV蛋白E6和E7的新蛋白质相互作用。62，63，64通过这种蛋白质组学技术，我的目标是进行E2结合伴侣的全面调查。

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Tan, a previous member of the Howley lab, have identified an uncharacterized interaction of BPV E2 with SMC5 and SMC6 (unpublished).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the interaction of HPV-16 and HPV-8 E2 with SMC6 through mammalian cell culture techniques and western blotting (unpublished).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