Regulation and Targeting of HIV-1 Integrase-RNA Interactions

摘要 人类免疫缺陷病毒1型(HIV-1)耐药变异株的出现和缺乏 一种有效的疫苗需要开发新型抗逆转录病毒药物。HIV-1的催化活性 整合酶(IN)已经成功地被几个高效和耐受性良好的链转移靶向 抑制剂(INSTI)。然而，尽管第二代INSTI的障碍很高，但突变带来了 在临床环境中已经报告了对多种INSTI的耐药性。因此，通过另一种选择来瞄准IN 机制可以补充现有的治疗策略，并大幅增加屏障，以 在INSTI治疗中出现耐药的HIV-1变种。 IN在HIV-1的生命周期中具有神秘的非催化功能，这一点早已为人所知。一定的 IN中的突变，统称为II类突变，据报道是多效性的，并导致 病毒颗粒组装、成熟和逆转录。在一项范式转换的研究中，我们发现 HIV-1IN以病毒粒子的形式与病毒RNA基因组(GRNA)结合，这种相互作用对 准确的病毒粒子形态发生。变构整合酶抑制剂对IN-gRNA相互作用的抑制 (Allini)或II类In取代导致与gRNA IS一起形成异常的“偏心”粒子 错位于空衣壳(CA)格子和病毒包膜之间。此外，我们已经证明，在 四聚化是RNA结合的关键，许多II类IN取代位于整个IN 通过调节IN四聚化抑制RNA结合。最后，我们实验室的工作证明了 病毒粒子中gRNA的过早降解及其与逆转录酶的物理分离 靶细胞中偏心颗粒的逆转录缺陷。重要的是，这种不合时宜的gRNA 降解不是由于抑制IN-gRNA相互作用本身，而是由于失去了与IN-gRNA相互作用的保护 在CA失稳时观察到类似的结果，CA晶格。总而言之，这些研究巩固了 IN-gRNA相互作用在病毒粒子成熟中的作用和论证了CA晶格在 靶细胞中病毒核酸的保护。 基于这些新的发现和广泛的初步数据，我们建议阐明 HIV-1 IN-gRNA相互作用的规则，IN如何与gRNA结合介导HIV-1衣壳的正确组装 晶格以及感染细胞如何感知和响应因抑制IN-gRNA而产生的异常颗粒 CA晶格的相互作用和失稳。这些研究将填补我们对 HIV-1 IN在颗粒成熟过程中的临界非催化作用及其抑制的后果 互动。总而言之，这个项目不仅将增强我们对艾滋病毒-1复制的基本知识，而且还将有助于 开发可在临床环境中补充基于INSTI的治疗的新型抗病毒药物。

Abstract The emergence of drug resistant human immunodeficiency virus type-1 (HIV-1) variants and the lack of an effective vaccine require the development of novel anti-retroviral drugs. The catalytic activity of HIV-1 integrase (IN) has been successfully targeted by several highly effective and well tolerated IN strand transfer inhibitors (INSTIs). However, despite high barriers with the second-generation INSTIs, mutations conferring resistance to multiple INSTIs have been reported in clinical settings. Thus, targeting IN through an alternative mechanism can complement the existing therapeutic strategies and substantially increase the barrier to emergence of drug resistant HIV-1 variants upon INSTI treatment. IN has long been known to have an enigmatic non-catalytic function in the HIV-1 life cycle. Certain mutations in IN, collectively referred to as class II mutations, are reportedly pleiotropic and result in defects in viral particle assembly, maturation and reverse transcription. In a paradigm-shifting study, we have discovered that HIV-1 IN binds to the viral RNA genome (gRNA) in virions and that this interaction is critically important for accurate virion morphogenesis. Inhibition of IN-gRNA interactions through allosteric integrase inhibitors (ALLINIs) or class II IN substitutions results in the formation of aberrant “eccentric” particles with the gRNA is mislocalized between the empty capsid (CA) lattice and the viral envelope. Furthermore, we have shown that IN tetramerization is critical for RNA-binding and that a number of class II IN substitutions located throughout IN inhibit RNA binding through modulation of IN tetramerization. Finally, work from our lab demonstrated that premature degradation of the gRNA and its physical separation from the reverse transcriptase enzyme in virions underlies the reverse transcription defects of eccentric particles in target cells. Importantly, this untimely gRNA degradation is not due to inhibition of IN-gRNA interactions per se, but rather due to loss of protection with the CA lattice, as a similar outcome was observed upon CA destabilization. Together, these studies cemented the role of IN-gRNA interactions in virion maturation and demonstrated the critical importance of the CA lattice in protection of viral nucleic acids in target cells. Based on these novel findings and extensive preliminary data, we propose to elucidate the nature and rules of HIV-1 IN-gRNA interactions, how IN binding to the gRNA mediates proper assembly of the HIV-1 capsid lattice and how infected cells sense and respond to aberrant particles generated upon inhibition of IN-gRNA interactions and destabilization of the CA lattice. These studies will fill a critical gap in our understanding of the critical noncatalytic function of HIV-1 IN in particle maturation and the consequences of inhibiting these interactions. Together, this project will not only enhance our basic knowledge of HIV-1 replication but also aid in the development of novel antivirals that can complement INSTI-based therapies in clinical settings.