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论的看法 HIV DNA整合的基础。直到最近，普遍的观点是整合酶四聚体，起作用 在内含体核蛋白复合体的背景下，催化逆转录病毒整合到染色质中。然而， 在过去的一年里，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 部分由这笔赠款资助的研究表明，整合酶八聚体促进了β-逆转录病毒的整合。更多 最近，冷冻电子显微镜揭示了HIV-1链转移复合体的结构，最终 整合途径中的端粒体复合体是多态的，包含两个简单的四聚体排列 以及更高阶的十二聚体/十六聚体。此外，高阶复数的形成依赖于 共同整合辅助因子LEDGF的整合酶结合域的存在。这些观察结果导致了 该领域的几个新问题，这些问题将在这次赠款申请中得到解决。例如，做intasome 链转移复合体之前的复合体也包括不同多聚体的混合物，或通过其 自然，整合途径的成熟是否需要更高阶多聚体的形成？几个尖端 包括单粒子低温电子显微镜和单分子荧光成像在内的方法将 被用来表征肠小体组装的机制基础，并随着复合体的成熟而发挥作用 沿着HIV-1整合途径。除了评估LEDGF在途径成熟中的作用外， LEDGF的结构将被确定为与核小体结合，并作为连接连接体与 核小体。除了引导整合到活性基因之外，LEDGF最近还参与了 艾滋病毒潜伏期的调控。尽管衣壳结合CPSF6因子比LEDGF发挥更大作用 与活性染色质的整合，CPSF6在调节HIV潜伏期中的潜在作用尚不清楚。在这里，我们将 全面解决两个主要的针对辅因子的整合，LEDGF和CPSF6在 HIV-1潜伏期在细胞系模型和原代T细胞中的建立和调节。完成 该项目将提供艾滋病毒-1整合到染色质的结构基础以及 在艾滋病毒前病毒潜伏期的建立和调控上扰乱这些途径。

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.