Reactivation of Innate Immune Response Pathways as Treatment for HIV Infection

重新激活先天免疫反应途径作为 HIV 感染的治疗方法

• 批准号: 9204184
• 负责人: Robyn Midori Kaake
• 金额: $ 5.61万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9204184
• 关键词: Affinity Chromatography; Antiviral Agents; Attenuated; Binding; CBFB gene; CRISPR/Cas technology; Cataloging; Catalogs; Cells; Communicable Diseases; Complex; Crystallography; Cytidine Deaminase; Data; Degradation Pathway; Electron Microscopy; Elements; Event; Failure; Gene Deletion; Genetic; Goals; HIV; HIV Infections; HIV therapy; HIV-1; Hybrids; Immune; Immune response; Infection; Knock-in; Knowledge; Ligase; Maps; Mass Spectrum Analysis; Mediating; Methodology; Methods; Mission; Modeling; Molecular; Molecular Conformation; Molecular Machines; Mutation; Natural Immunity; Outcome; Pathway interactions; Process; Protein-Protein Interaction Map; Proteins; Proteomics; Public Health; Research; Structural Models; Structure; System; Technology; Testing; Therapeutic; Therapeutic Intervention; Ubiquitin; Viral Genome; Virus; Work; base; cofactor; crosslink; improved; in vivo; meetings; multicatalytic endopeptidase complex; non-Native; novel; pathogen; protein complex; research study; restoration; tool; treatment strategy; ubiquitin-protein ligase; vif Gene Products; virus host interaction

PROJECT SUMMARY/ABSTRACT Cellular APOBEC3 proteins are potent HIV-1 restriction factors that catalyze the lethal hypermutation of viral genomes. HIV-1 protein Vif deactivates this restriction process by targeting APOBEC3 proteins for ubiquitylation and proteasomal degradation. Reactivation of APOBEC3 innate immune response pathways is a promising HIV-1 therapeutic strategy. Consequently, there is a critical need to identify key regulatory and structural components important for HIV-1 Vif-mediated destruction of cellular APOBEC3 restriction factors. The long-term goal of this proposal is to establish integrative proteomic and structural technologies for determination of virus-host interactions, especially in the context of HIV evasion of innate immune pathways. The next step to attaining this goal, and overall objective of this proposal, is to identify protein components and structural features of the Vif-APOBEC3 pathway that can be manipulated to protect APOBEC3 proteins from Vif-induced degradation. This objective will be reached by testing the central hypothesis that host-host protein interactions of the ubiquitin-proteasome system can be modulated to reactivate APOBEC3 restriction against HIV-1. To test this hypothesis the following three specific aims will be pursued. (Aim1) Identify candidate regulators of Vif-dependent APOBEC3 degradation. This first aim will be carried out by in vivo cross-linking and affinity purification mass spectrometry methodologies in combination with protein-protein interaction mapping analyses. (Aim2) Identify structural features and subunits essential to APOBEC3-Vif- CRL5 complex stability and function. Structural models of APOBEC3-Vif-CRL5 complexes will be generated using the integrative modeling platform which will merge solved structures (from crystallography, electron microscopy, SAX, etc.), with distance constraints and protein interaction interface data collected from structural cross-linking mass spectrometry experiments. (Aim3) Interrupt Vif-APOBEC3 ubiquitylation and degradation pathways to reactivate APOBEC3 restriction against HIV-1 infection. This will be accomplished using CRISPR/Cas9 gene deletion or “knock-in” methods that will introduce protein interaction mutations into immunological cells to test their restrictive potential against HIV infection. Successful completion of the proposed research is expected to expand understanding of the molecular details involved in HIV-1 infection and evasion of innate immunity, as well as host response to retroviral pathogens. This will be a significant contribution as it will highlight new targets for improved therapeutic strategies for treatment of HIV-1 infection.

项目摘要/摘要 细胞APOBEC3蛋白是潜在的HIV-1限制因子 基因组。 HIV-1蛋白VIF通过靶向apobec3蛋白来停用了这种限制过程 泛素化和蛋白酶体降解。 APOBEC3先天免疫响应途径的重新激活是 有希望的HIV-1治疗策略。因此，肯定需要确定关键监管和 对于HIV-1 VIF介导的细胞APOBEC3限制因子的破坏至关重要的结构成分。 该提案的长期目标是建立综合蛋白质组学和结构技术 确定病毒宿主相互作用，尤其是在先天免疫途径的HIV演化的背景下。 实现此目标的下一步以及该提案的总体目标是确定蛋白质成分 以及可以操纵以保护APOBEC3蛋白的VIF-APOBEC3途径的结构特征 来自VIF引起的降解。通过测试宿主主持人的中心假设来实现此目标 泛素 - 蛋白酶体系统的蛋白质相互作用可以调节以重新激活APOBEC3限制 反对HIV-1。为了检验该假设，将追求以下三个特定目标。 （AIM1）识别 依赖VIF的APOBEC3降解的候选调节剂。第一个目标将由体内实施 交联和亲和力纯化质谱法结合蛋白质蛋白 相互作用映射分析。 （AIM2）确定APOBEC3-VIF-必不可少的结构特征和亚基 CRL5复合稳定性和功能。将生成APOBEC3-VIF-CRL5复合物的结构模型 使用将合并解决结构的集成建模平台（来自晶体学，电子 显微镜，SAX等），具有从距离限制和蛋白质相互作用接口数据收集的 结构交联质谱实验。 （AIM3）中断VIF-APOBEC3泛素化和 降解途径可重新激活APOBEC3限制HIV-1感染。这将完成 使用CRISPR/CAS9基因缺失或将蛋白质相互作用突变引入到 免疫细胞测试其针对HIV感染的限制潜力。成功完成 拟议的研究有望扩大对HIV-1感染涉及的分子细节的理解 和先天免疫的进化以及对逆转录病毒病原体的宿主反应。这将是重要的 贡献将重点介绍改善治疗HIV-1治疗策略的新目标 感染。