HIV-host interactions driving virus integration

HIV-宿主相互作用驱动病毒整合

• 批准号: 10242908
• 负责人: Alan N. Engelman
• 金额: $ 44.85万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242908
• 关键词: Address; Affinity; Applications Grants; Automobile Driving; Binding; Biochemical; CRISPR/Cas technology; Capsid; Cell Line; Cell model; Cells; Chromatin; Chromatin Structure; Complex; Consensus; Cryoelectron Microscopy; DNA; DNA Integration; Epigenetic Process; Evolution; Fluorescence; Funding; Genes; Goals; Grant; HIV; HIV Infections; HIV-1; Human; Image; In Vitro; Integrase; Integration Host Factors; Knock-out; Lead; Link; Mediating; Methods; Modeling; Monitor; Mouse Mammary Tumor Virus; Nature; Nucleoproteins; Nucleosomes; Paper; Pathway interactions; Play; Process; Proteins; Proviruses; RNA; Regulation; Retroviridae; Role; Seminal; Series; Structure; T-Lymphocyte; Technology; Time; Viral; Virus Integration; Virus Latency; Work; cofactor; fluorescence imaging; particle; preference; reactivation from latency; response; single molecule; stoichiometry; structural biology; three dimensional structure; tool; viral DNA

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.

抽象的 HIVE 中心和其他机构的研究结果改变了我们思考机制的方式 HIV DNA 整合的基础。直到最近，人们一致认为整合酶四聚体可以发挥作用 在嵌体核蛋白复合物的背景下，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 这笔赠款的部分资助表明，β-逆转录病毒整合是由整合酶八聚体促进的。更多的 最近，冷冻电子显微镜揭示了 HIV-1 链转移复合物的结构，即最终的 整合途径中的嵌体复合物是多态性的，包含简单的四聚体排列 以及高阶十二聚体/十六聚体。高阶复杂的形成此外还取决于 共同整合辅因子 LEDGF 的整合酶结合域的存在。这些观察导致 该领域的几个新问题将在本次赠款申请中得到解决。例如，做 intasome 链转移复合物之前的复合物还包含不同多聚体的混合物，或者通过其 本质上，整合途径的成熟是否需要高阶多聚体的形成？几条尖端 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 用于表征复合体成熟时整合体组装和功能的机制基础 沿着 HIV-1 整合途径。除了评估 LEDGF 在通路成熟中的作用外， LEDGF 结构将被确定为与核小体结合，并且作为连接内体与核小体的系链 核小体。除了指导整合到活性基因之外，LEDGF 最近还与 HIV潜伏期的调节。尽管衣壳结合 CPSF6 因子比 LEDGF 发挥更大的作用来指导 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 全面阐述两个主要整合靶向辅助因子 LEDGF 和 CPSF6 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其后果 破坏这些途径对艾滋病毒原病毒潜伏期的建立和调节。