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

基本信息

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
项目类别：
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

项目摘要

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Tan, a previous member of the Howley lab, have identified an uncharacterized interaction of BPV E2 with SMC5 and SMC6 (unpublished).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the interaction of HPV-16 and HPV-8 E2 with SMC6 through mammalian cell culture techniques and western blotting (unpublished).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）：Howley 实验室前成员 Alvin Tan 进行的牛乳头瘤病毒 (BPV) MS/ComPASS 实验的初步数据已确定 BPV E2 与 SMC5 和 SMC6 之间存在未表征的相互作用（未发表）。 SMC5/6 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，并与 E2 一起导致双链断裂。18,19,20,56 E1-E2 复制焦点包含 DDR 蛋白和同源复制标记。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或复制焦点定位到 DNA 的脆弱点中发挥作用，这些脆弱点更容易受到病毒 DNA 的整合。我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。 Beta 和 Gamma 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，与染色质的宿主结合伴侣未知。14,35,39 HPV-8 E2和 SMC5/6 具有相同的独特染色体关联（与着丝粒和 rDNA 位点），因此我的目的是确定 SMC5/6 复合体是否是主要的这些 E2 蛋白的束缚因子。48,50,54,65 在基因组维持阶段建立这种复合物的束缚作用，支持了以下假设：在诱导宿主 DDR，发生在基因组扩增阶段。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，并确定致癌性和低风险 HPV 的病毒-宿主蛋白相互作用之间的差异。我还致力于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。我计划对多种 E2 蛋白进行蛋白质组筛选。该技术将包括 E2 相关蛋白复合物的质谱 (MS)，该复合物将从稳定表达这些蛋白的细胞系中进行免疫共沉淀，以及随后与哈佛大学 Harper 实验室合作进行的比较蛋白质组分析软件套件 (ComPASS) 分析医学院。该技术有望识别特定 HPV 属或种所独有的或在所有 HPV 类型中保守的新型 E2 高可信度相互作用蛋白。这些技术已在 Howley 实验室之前的研究中成功使用，以揭示 HPV 蛋白 E6 和 E7 的新型蛋白质相互作用。62,63,64 通过这种蛋白质组学技术，我的目标是对 E2 结合伴侣进行全面调查。