Structural determinants for integrase pleiotropism in viral maturation

病毒成熟过程中整合酶多效性的结构决定因素

• 批准号: 9411652
• 负责人: Mamuka Kvaratskhelia
• 金额: $ 85.73万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9411652
• 关键词: Acylation; Antiviral Agents; Binding; Biochemical; Biological; Biological Process; Biology; Capsid; Clinical Trials; Collaborations; Complementary DNA; Complex; Computer Simulation; Coupled; Drug resistance; Electron Microscopy; Electrons; Elements; Ensure; Event; Evolution; Fluorescence; Free Energy; Funding; Genome; HIV; HIV-1; HIV-1 integrase; Human Chromosomes; Hydroxyl Radical; Image; Impairment; Infection; Integrase; Integrase Inhibitors; Length; Microscopy; Modeling; Molecular; Nature; Nucleocapsid; Phenotype; Play; Primer Extension; RNA; Research; Research Personnel; Retroviridae; Ribonucleoproteins; Role; Scientist; Seminal; Shapes; Site-Directed Mutagenesis; Structure; Technology; Viral; Viral Proteins; Virion; Virus Integration; crosslinking and immunoprecipitation sequencing; experimental study; inhibitor/antagonist; innovation; particle; pleiotropism; resistance mutation; single molecule; small molecule; solid state nuclear magnetic resonance; therapeutic target; viral DNA; viral RNA

Abstract The studies in Project 1 will center on elucidating structural determinants for a second, non-catalytic function of HIV-1 integrase (IN) during virion maturation. While an essential role of IN for integration of viral cDNA into human chromosome during early steps of HIV-1 infection has been long established and successfully exploited as a therapeutic target, the recent studies with allosteric IN inhibitors (ALLINIs) by HIVE investigators coupled with earlier site-directed mutagenesis experiments have suggested that IN plays an active role in viral particle maturation. During the past funding period our studies with ALLINIs, a new class of very promising antiviral compounds, which are currently in clinical trials, have uncovered that these inhibitors induce hyper or aberrant multimerization of HIV-1 IN and yield eccentric, non-infectious virions where the ribonucleoprotein complexes (RNPs) are mislocalized outside of the translucent capsid cores. Subsequent collaborative efforts by HIVE and CRNA investigators have revealed that IN binds the viral RNA genome to ensure correct localization of RNPs within the protective capsid core, whereas ALLINI treatments impair IN-RNA interactions and result in the eccentric core phenotype. These seminal findings will now be extended to elucidate key structural interactions that underlie these biological events. In particular, our experiments will take advantage of cutting-edge technologies available within the HIVE center as well as utilize complementary expertise of CRNA and PCHPI scientists to accomplish the following aims. Aim 1 will determine the cryo-EM structure of IN bound to a cognate RNA element; aim 2 will dissect complementary interactions of IN and nucleocapsid with viral RNA using single molecule fluorescence and SHAPE; aim 3 will examine the significance of IN-RNA interactions in non-primate lentiviruses and other retroviruses using CLIP-seq and biochemical approaches; aim 4 will investigate the structural basis for ALLINI induced hyper-multimerization of full length wild type IN using solid state NMR and free energy calculations; and aim 5 will generate mesoscale models of correctly matured and ALLINI treated eccentric virus particles. These highly innovative experiments will push the boundaries of rapidly developing technologies such as single particle cryo-EM, solid-state NMR and computational modeling as well as critically advance our understanding of molecular events that shape the formation of infectious virions.

摘要 项目1的研究将集中在阐明第二种非催化功能的结构决定因素上， HIV-1整合酶（IN）在病毒体成熟过程中的作用。虽然IN对于病毒cDNA整合到 人类染色体在HIV-1感染的早期阶段已经建立并成功地利用 作为治疗靶点，HIVE研究人员最近对变构IN抑制剂（ALLINI）的研究， 早期的定点突变实验表明，IN在病毒颗粒中起着积极的作用， 成熟在过去的资助期间，我们对ALLINI的研究，这是一类非常有前途的新型抗病毒药物， 目前正在临床试验中的化合物已经发现，这些抑制剂可以诱导过度或异常的 多聚化的HIV-1 IN和产生偏心，非感染性病毒粒子，其中核糖核蛋白复合物 RNP被错误定位在半透明衣壳核心的外部。HIVE随后的合作努力， CRNA研究人员揭示，IN结合病毒RNA基因组，以确保RNP的正确定位 在保护性衣壳核心内，而ALLINI治疗损害IN-RNA相互作用并导致 偏心核表型这些开创性的发现现在将被扩展以阐明关键的结构相互作用 这些生物学事件的基础。特别是，我们的实验将利用最先进的 HIVE中心内可用的技术以及利用CRNA和PCHPI的互补专业知识 科学家要实现以下目标。目的1将确定与同源物结合的IN的冷冻电镜结构 RNA元件;目的2将使用单个RNA元件来剖析IN和核衣壳与病毒RNA的互补相互作用。 分子荧光和SHAPE;目的3将检查非灵长类动物中IN-RNA相互作用的意义 慢病毒和其他逆转录病毒使用CLIP-seq和生物化学方法;目的4将研究 使用固态NMR，ALLINI诱导全长野生型IN超多聚化的结构基础， 自由能计算; aim 5将生成正确成熟和ALLINI处理的中尺度模型 偏心病毒颗粒这些高度创新的实验将推动快速发展的边界 技术，如单粒子cryo-EM，固态NMR和计算建模以及关键 推进我们对形成传染性病毒体的分子事件的理解。