REGULATION AND TARGETING OF HIV-1 INTEGRASE-RNA INTERACTIONS

摘要 基于二十年前的突变研究，HIV-1整合酶(IN)蛋白被提出在 在艾滋病毒-1复制的后期阶段发挥作用。这些突变统称为II类突变， 对病毒复制的多个步骤产生不利影响，包括颗粒组装、成熟和随后的 逆转录。一些II类突变特别地损害了颗粒成熟，导致了 病毒核糖核蛋白复合体(RNPs)错误定位于病毒核糖核蛋白复合体外的异常病毒核心 圆锥形衣壳核心。然而，近20年来，IN如何能够做出贡献一直是个谜 适当的病毒颗粒成熟。 变构蛋白抑制剂(ALLINI)是最近出现的一类有前途的新型抗逆转录病毒药物 精选的化合物目前正在进行临床试验。尽管ALLINI最初的设计目的是阻止 IN与其细胞辅因子LEDGF/p75的相互作用，最近被证明它们有效地损害了 HIV-1复制的后期步骤。类似于某些II类IN突变，这些抑制物选择性地干扰 适当的病毒颗粒成熟，产生偏心定位的非传染性颗粒。虽然 已有研究表明，AlLINI可以促进多聚体的异常，这一事件是如何不利地导致 衣壳核外的RNPs的错误定位仍然未知。 基于这些观察，我们最近探索了 IN可能结合病毒的可能性耐人寻味 成熟颗粒中的RNA基因组以及Allini可能干扰IN-RNA相互作用 粒子形成。 为了验证这一假设，我们使用了尖端的CLIP-SEQ(交联型-SEQ)。 免疫沉淀-测序)方法和互补的生化方法。这些研究 首次揭示了IN与病毒RNA基因组上的特定序列结合，并且某些 IN和ALLINI内的突变有效地阻止了这些相互作用。我们打算继续我们的学业。 确定等位基因影响IN-RNA相互作用的机制以及IN-RNA是如何 相互作用调节着颗粒的成熟。我们还建议使用异常形成的偏心堆芯 在存在ALLINI/II类突变的情况下产生的基因，作为了解早期进入后事件的工具 感染。因此，该项目不仅将提供前所未有的洞察力，了解HIV-1在人类免疫系统中的新作用 颗粒组装和ALLINI的主要作用机制，但也将促进发展 了解HIV-1复制的早期进入后事件的研究。

Abstract Based on mutational studies two decades ago, HIV-1 Integrase (IN) protein has been proposed to play a role in late stages of HIV-1 replication. These mutations, collectively referred to as Class II mutations, adversely affect multiple steps of virus replication including particle assembly, maturation and subsequent reverse transcription. Some class II mutations specifically impair particle maturation leading to the formation of aberrant viral cores in which the viral ribonucleoprotein complexes (RNPs) are mislocalized outside of the conical capsid core. However, for nearly 20 years it has remained enigmatic as to how IN can contribute to proper viral particle maturation. Allosteric IN inhibitors (ALLINIs) have recently emerged as a promising new class of antiretroviral agents and select compounds are currently in clinical trials. Although ALLINIs were initially designed to block the interaction of IN with its cellular cofactor LEDGF/p75, it has recently been shown that they potently impair the late steps of HIV-1 replication. Similar to certain Class II IN mutations, these inhibitors selectively interfere with proper virus particle maturation and yield non-infectious particles with eccentrically positioned RNPs. Although it has been shown that ALLINIs can promote aberrant IN multimerization, how this event adversely results in the mislocalization of the RNPs outside the capsid core has remained unknown. Based on these observations, we have recently explored the intriguing possibility that IN may bind the viral RNA genome in mature particles and that ALLINIs may interfere with IN-RNA interactions critical for proper particle formation. To test this hypothesis, we have employed the cutting-edge CLIP-seq (crosslinking- immunoprecipitation-sequencing) methodology and complementary biochemical approaches. These studies have revealed for the first time that IN binds to specific sequences on the viral RNA genome and that certain mutations within IN and ALLINIs potently block these interactions. We propose to continue our studies in identifying the details of the mechanism by which ALLINIs affect IN-RNA interactions and how IN-RNA interactions regulate particle maturation. We also propose to use the aberrantly formed eccentric cores generated in the presence of ALLINIs/Class II mutations as a tool to understand the early post-entry events in infection. As such, this project will not only provide unprecedented insight into the novel role of HIV-1 IN in particle assembly and the primary mechanism of action of ALLINIs, but will also facilitate the development of studies to understand early post-entry events in HIV-1 replication.