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 Our published and unpublished results suggest that a host factor – the transmembrane protease γ-secretase – is endowed with a novel chaperone activity that promotes insertion of an HPV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 这是一项新的多PI拨款的提议，涉及两名高级调查人员，具有互补的 研究人类乳头瘤病毒(HPV)感染中一个重要但知之甚少的步骤的专业知识。人类乳头瘤病毒 对全球约5%的人类癌症负有责任。在感染期间，HPV被内化为 内体从其被定位到逆行途径以运输到TGN的途径 生殖性感染的核心。内小体到高尔基体靶向代表已承诺的感染 步骤，并且仍然知之甚少。我们已发表和未发表的结果表明，一个宿主因素-- 跨膜蛋白水解酶γ-分泌酶-被赋予一种新的伴侣活性，促进 将一种称为L2的HPV衣壳蛋白插入到内吞体膜中。L2的膜插入 随后向细胞质的突起招募胞浆宿主成分，将HPV传递到 高尔基。值得注意的是，我们还发现，人乳头瘤病毒感染稳定了多亚单位γ-分泌酶复合体。 因此，这项提议的中心目标是在分子水平上阐明γ是如何- 分泌酶促进L2膜插入及人乳头瘤病毒如何调节γ-分泌酶的功能 协同推进人乳头瘤病毒感染。我们将鉴定将γ分泌酶招募到人乳头状瘤病毒的细胞蛋白， 确定γ分泌酶介导的衣壳重排是否允许L2从衣壳中出现，以及 尝试在体外重建膜插入并测定相关的γ-分泌酶亚基和 活动。我们将确定γ-分泌酶是否在细胞穿透性多肽的插入中发挥作用，或者 总体上是逆行贩卖。我们将研究N末端跨膜结构域在L2中的作用 并试图开发不依赖γ分泌酶的病毒突变体。最后，我们 将确定人乳头瘤病毒感染如何稳定γ-分泌酶复合体，确定与此相关的其他因素 过程，并确定L2跨膜结构域是否足以稳定和膜 插入。综上所述，这些实验将为γ-分泌酶在体内的不寻常作用提供新的线索 HPV的进入，提高了我们对HPV感染这一关键步骤的认识，阐明了HPV感染的新方面 普通细胞生物学，并可能为预防感染的新治疗方法指明方向 这些重要的人类病原体。