HIV-host interactions driving virus integration

摘要 来自蜂巢中心和其他人的结果已经改变了我们对机械的思考方式， HIV DNA整合的基础。直到最近，一致的观点是整合酶四聚体， 在整合体核蛋白复合物的情况下，催化逆转录病毒整合到染色质中。然而，在这方面， 在过去一年中，以四聚体为中心的逆转录病毒整合观点被暴露为过于简单化。工作 部分由该赠款资助的研究表明，β-逆转录病毒的整合是由整合酶八聚体促进的。更 最近，冷冻电子显微镜显示，HIV-1链转移复合物的结构，最终的 整合途径中的整合体复合物是多态的，包含简单的四聚体排列， 以及更高级的十二聚体/十六聚体。此外，更高级的复合物的形成取决于 共同整合辅因子LEDGF的整合酶结合结构域的存在。这些观察导致 该领域的几个新问题，这将在本赠款申请中得到解决。例如，do intasome 在链转移复合物之前的复合物也包含不同多聚体的混合物，或者，通过其 自然界中，整合途径的成熟是否需要高阶多聚体的形成？几个切削刃 包括单粒子冷冻电子显微镜和单分子荧光成像在内的方法将 用于表征整合体组装的机制基础，并随着复合物成熟而发挥作用 沿着HIV-1整合途径。除了评估LEDGF在途径成熟中的作用外， LEDGF结构将被确定为与核小体结合，并且作为将整合体连接到核小体的系链。 核小体除了指导整合到活性基因中之外，LEDGF最近还被牵连到基因组中。 调节HIV潜伏期。尽管衣壳结合CPSF 6因子比LEDGF在指导细胞凋亡中发挥更大的作用， 尽管CPSF 6与活性染色质整合，但CPSF 6在调节HIV潜伏期中的潜在作用是未知的。在这里，我们将 全面解决两个主要的整合靶向辅因子，LEDGF和CPSF 6的作用， 在细胞系模型以及原代T细胞中HIV-1潜伏期的建立和调节。完成 该项目将提供HIV-1整合到染色质中的结构基础以及 破坏这些通路对HIV前病毒潜伏期的建立和调节。

Abstract Results from the HIVE Center and from others have transformed the way that we think about the mechanistic basis of HIV DNA integration. Until quite recently, the consensus view was that an integrase tetramer, working in the context of the intasome nucleoprotein complex, catalyzed retroviral integration into chromatin. However, over the past year, the tetramer-centric view of retroviral integration has been exposed as overly simplistic. Work in part funded by this grant revealed that beta-retroviral integration is promoted by an integrase octamer. More recently, cryo-electron microscopy revealed that the structure of the HIV-1 strand transfer complex, the final intasome complex in the integration pathway, is polymorphic, containing both simple tetramer arrangements as well as higher-order dodecamers/hexadecamers. Higher-order complex formation moreover depended on the presence of the integrase-binding domain of the common integration co-factor LEDGF. These observations lead to several new questions in the field, which will be addressed in this grant application. For example, do intasome complexes that precede the strand transfer complex also comprise a mixture of different multimers, or, by its nature, does integration pathway maturation necessitate higher-order multimer formation? Several cutting edge approaches, including single-particle cryo-electron microscopy and single-molecule fluorescence imaging, will be used to characterize the mechanistic basis of intasome assembly and function as the complexes mature along the HIV-1 integration pathway. In addition to assessing the role of LEDGF in pathway maturation, the LEDGF structure will be determined bound to nucleosomes, and as the tether that links the intasome to the nucleosome. In addition to directing integration into active genes, LEDGF has recently been implicated in the regulation of HIV latency. Although the capsid binding CPSF6 factor plays a greater role than LEDGF to direct integration to active chromatin, a potential role for CPSF6 in regulating HIV latency is unknown. Here, we will comprehensively address the roles of the two main integration targeting cofactors, LEDGF and CPSF6, in the establishment and regulation of HIV-1 latency in cell line models as well as in primary T cells. The completion of this project will provide for the structural basis of HIV-1 integration into chromatin and the consequences of disrupting these pathways on the establishment and regulation of HIV proviral latency.