Mechanism of gamma-secretase action during HPV infection

HPV感染过程中γ-分泌酶的作用机制

• 批准号: 10132235
• 负责人: Daniel C. Dimaio
• 金额: $ 60.8万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-24 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10132235
• 关键词: Address; Anogenital cancer; Antiviral Agents; Binding; Biochemical; Capsid; Capsid Proteins; Cell Nucleus; Cell physiology; Cells; Cellular biology; Complex; Cytoplasm; DNA Viruses; Endocytosis; Endosomes; Event; Foundations; Golgi Apparatus; Golgi Targeting; Grant; Human; Human Papillomavirus; Human papilloma virus infection; In Vitro; Individual; Infection; Infection prevention; Integral Membrane Protein; Integration Host Factors; L2 viral capsid protein; Light; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Mass Spectrum Analysis; Mediating; Membrane; Minor; Molecular; Molecular Chaperones; Molecular Conformation; Mutation; N-terminal; Pathway interactions; Peptide Hydrolases; Peptides; Play; Preventive vaccine; Process; Proteins; Proteomics; Publishing; Research Personnel; Role; Route; Testing; Transmembrane Domain; Vaccines; Viral; Viral Genome; Virion; Virus; base; burden of illness; combat; endosome membrane; experimental study; gamma secretase; high risk; human pathogen; improved; insight; intracellular protein transport; malignant oropharynx neoplasm; mutant; novel; novel therapeutic intervention; prevent; protein complex; receptor; reconstitution; recruit; trafficking; trans-Golgi Network; virology

Abstract This is a proposal for a new, multi-PI grant involving two senior investigators with complementary expertise to study an essential but poorly understood step in human papillomavirus (HPV) infection. HPV is responsible for approximately 5% of human cancer worldwide. During infection, HPV is internalized to the endosome from where it is targeted to the retrograde pathway for transport to the TGN en route to the nucleus for productive infection. Endosome-to-Golgi targeting represents the committed infection step and remains poorly understood. Our published and unpublished results suggest that a host factor – the transmembrane protease γ-secretase – is endowed with a novel chaperone activity that promotes insertion of an HPV capsid protein called L2 into the endosome membrane. Membrane insertion of L2 and subsequent protrusion into the cytoplasm recruits cytosolic host components that deliver HPV to the Golgi. Strikingly, we also found that HPV infection stabilizes the multi-subunit γ-secretase complex. Accordingly, the central objectives of this proposal are to elucidate, at the molecular level, how γ- secretase promotes membrane insertion of L2 and how HPV regulates the function of γ-secretase to coordinately promote HPV infection. We will identify cellular proteins that recruit γ-secretase to HPV, determine if γ-secretase-mediated capsid rearrangements allow L2 to emerge from the capsid, and attempt to reconstitute membrane insertion in vitro and determine the relevant γ-secretase subunits and activities. We will determine if γ-secretase plays a role in insertion of cell-penetrating peptides or retrograde trafficking in general. We will examine the role of the N-terminal transmembrane domain in L2 in these activities and attempt to develop viral mutants that are independent of γ-secretase. Finally, we will determine how HPV infection stabilizes the γ-secretase complex, identify other factors involved in this process, and determine if the L2 transmembrane domain is sufficient for stabilization and membrane insertion. Taken together, these experiments will shed new light on the unusual role of γ-secretase in HPV entry, improve our understanding of this crucial step in HPV infection, elucidate new aspects of general cell biology, and possibly point the way to new therapeutic approaches to prevent infection by these important human pathogens.

摘要 这是一个新的，多PI赠款涉及两名高级研究人员与互补的建议， 研究人乳头瘤病毒（HPV）感染中一个重要但知之甚少的步骤。HPV 导致了全球约5%的人类癌症。在感染过程中，HPV被内化， 内体从那里靶向逆行途径，转运到TGN， 生产性感染的核心。内体到高尔基体的靶向代表了确定的感染 步和仍然知之甚少。我们已发表和未发表的结果表明宿主因素- 跨膜蛋白酶γ-分泌酶-被赋予了一种新的伴侣活性， 将称为L2的HPV衣壳蛋白插入内涵体膜。L2膜插入 并且随后突出到细胞质中募集胞质宿主成分，其将HPV递送到细胞质中。 高尔基引人注目的是，我们还发现HPV感染使多亚基γ-分泌酶复合物稳定。 因此，本提案的中心目标是在分子水平上阐明γ- 分泌酶促进L2的膜插入以及HPV如何调节γ-分泌酶的功能， 协同促进HPV感染。我们将鉴定将γ-分泌酶募集到HPV的细胞蛋白， 确定γ-分泌酶介导的衣壳重排是否允许L2从衣壳中出现，和 尝试在体外重建膜插入并确定相关的γ-分泌酶亚基， 活动我们将确定γ-分泌酶是否在细胞穿透肽的插入中发挥作用， 一般的逆向贩运。我们将研究N-末端跨膜结构域在L2中的作用。 在这些活动中，并试图开发不依赖于γ-分泌酶的病毒突变体。最后我们 将确定HPV感染如何稳定γ-分泌酶复合体，确定参与此过程的其他因素， 处理，并确定L2跨膜结构域是否足以稳定和膜 插入。总之，这些实验将揭示γ-分泌酶的不寻常作用， HPV进入，提高我们对HPV感染中这一关键步骤的理解，阐明 一般细胞生物学，并可能指出新的治疗方法，以防止感染的方式， 这些重要的人类病原体。