Structural determinants for integrase pleiotropism in viral maturation

摘要 项目1中的研究将集中在阐明第二个非催化功能的结构决定因素 病毒粒子成熟过程中的HIV-1整合酶(IN)。而IN在整合病毒基因的过程中起着至关重要的作用 人类在HIV-1感染早期阶段的染色体早已被建立并成功地被利用 作为治疗的靶点，最近由蜂巢研究者联合进行的变构蛋白抑制剂(ALLINI)的研究 与早期的定点突变实验表明，IN在病毒颗粒中发挥着积极的作用 成熟。在过去的资助期间，我们对ALLINI进行了研究，这是一种非常有前途的新型抗病毒药物 目前正在进行临床试验的化合物已经发现，这些抑制剂会导致兴奋或异常。 HIV-1在体内的多聚体并产生偏心的非传染性病毒粒子，其中核糖核蛋白复合体 (RNP)错误地定位在半透明衣壳核心之外。HIVE和 CRNA研究人员发现，IN与病毒RNA基因组结合，以确保RNPs的正确定位 在保护性衣壳核心内，而Allini处理损害IN-RNA相互作用并导致 偏心的核心表型。这些开创性的发现现在将被扩展到阐明关键的结构相互作用 是这些生物事件的基础。特别是，我们的实验将利用尖端技术 蜂巢中心内可用的技术以及利用CRNA和PCHPI的互补专业知识 科学家们要完成以下目标。目标1将确定与同源词绑定的IN的冷冻-EM结构 RNA元素；Aim 2将使用单一的 分子荧光和形状；Aim 3将研究IN-RNA相互作用在非灵长类动物中的意义 慢病毒和其他逆转录病毒使用片段序列和生化方法；目标4将调查 Allini诱导的全长野生型超多聚体的固体核磁共振和结构基础 自由能计算；目标5将生成正确成熟和Allini处理的中尺度模式 偏心的病毒颗粒。这些极具创新性的实验将推动快速发展的 单粒子冷冻-EM、固态核磁共振和计算建模等技术以及关键 促进我们对塑造传染性病毒粒子形成的分子事件的理解。

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.