Structural Control of Human Co-factors for Retroviral Gene Expression

逆转录病毒基因表达的人类辅因子的结构控制

• 批准号: 9008671
• 负责人: CLARA KIELKOPF
• 金额: $ 29.55万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9008671
• 关键词: Address; Affinity; Alternative Splicing; Binding; Binding Sites; Biochemical; Biochemistry; Biological Assay; Biology; Calorimetry; Cell Extracts; Cell physiology; Cells; Collaborations; Complex; Crystallization; Development; Drug resistance; Gene Expression; Genome; Genomics; HIV; HIV-1; Hand; Homologous Gene; Housing; Human; Human Cell Line; Immune; Infection; Integration Host Factors; Knowledge; Laboratories; Length; Life Cycle Stages; Ligands; Mediating; Medicine; Messenger RNA; Molecular; Molecular Biology; Mutation; Nucleocapsid Proteins; Pathway interactions; Pharmaceutical Preparations; Phase; Plague; Process; Production; Proteins; RNA; RNA Binding; RNA Recognition Motif; RNA Splicing; Resistance; Resolution; Retroviridae; Retrovirology; Role; SF1; Small Interfering RNA; Specificity; Spliceosomes; Staging; Structural Biochemistry; Structure; Surface; Testing; Therapeutic; Therapeutic Intervention; Titrations; Transcript; U2 Small Nuclear Ribonucleoprotein; U2 small nuclear RNA; Universities; Ursidae Family; Variant; Viral; Virus; Virus Replication; Work; X-Ray Crystallography; Yeasts; base; biochemical tools; cofactor; genomic RNA; insight; interdisciplinary approach; knock-down; mRNA Precursor; macromolecule; medical schools; novel; novel therapeutic intervention; protein expression; public health relevance; research study; rev Protein; small hairpin RNA; small molecule; stem; structural biology; therapeutic development; virology; virus host interaction

 DESCRIPTION (provided by applicant): In this project, we will dissect the activity of key host cofactor Tat-SF1 in human versus HIV-1 RNA splicing. Complex retroviruses such as HIV-1 co-opt the human spliceosome machinery for tightly coordinated production of their spliced mRNAs and genomic RNAs during the early and late stages of the viral life cycle. At present, the molecular mechanisms responsible for the dominant progression of HIV-1 through the host splicing cycle remain outstanding questions in the field. Tat-SF1 is a host protein that is critica in the production of fully spliced HIV-1 mRNAs in the early stage of the retroviral life cycle. Tat SF1 is known to associate with the U2 small nuclear ribonucleoprotein subunit (snRNP) of the spliceosome and regulate the splicing of specific human transcripts. As such, studies of Tat-SF1 will be important to understand host-virus interactions, thereby laying the groundwork for the development of new biochemical tools or therapeutic intervention approaches at the level of splicing. At present, the underlying structural and functional roles of Tat-SF1 in HIV-1 and human splicing remain unknown. We will address this knowledge gap by a strong collaboration involving multidisciplinary approaches that include structural biochemistry of pre-mRNA splicing factors, molecular biology, and virology. Specifically, we will determine Tat- SF1 interactions among the human U2 snRNA and U2 snRNP subunits, and test these interfaces for functions in human splicing versus HIV-1 splicing and infectivity. We also will investigate a new mechanism of action for HIV-1 to pre-empt the human splicing machinery, in which the early stage HIV-1 Rev protein promotes late stage unspliced HIV-1 RNAs by disrupting the host Tat-SF1-U2 snRNP complex. Already we have: (i) produced diffracting crystals of the Tat-SF1 RNA recognition motif, (ii) obtained an initial structure of a complex between the Tat-SF1 and the SF3b155 subunit of the U2 snRNP, and (iii) generated a stable knockdown of Tat-SF1 in a human cell line. Moreover, our preliminary results demonstrate that the HIV- 1 Rev protein associates with human Tat-SF1. In addition to elucidating new mechanisms of action for HIV-1 to manipulate host pathways of gene expression, successful completion of the proposed experiments will set the stage for new therapeutic strategies that target a host cofactor, thereby circumventing drug resistance challenges that plague existing anti-HIV-1 treatments.

 描述（由申请人提供）：在本项目中，我们将剖析关键宿主辅助因子 Tat-SF1 在人类与 HIV-1 RNA 剪接中的活性。 HIV-1 等复杂的逆转录病毒在病毒生命周期的早期和晚期阶段，利用人类剪接体机制来紧密协调生产其剪接 mRNA 和基因组 RNA。目前，HIV-1通过宿主剪接周期的主要进展的分子机制仍然是该领域的悬而未决的问题。 Tat-SF1 是一种宿主蛋白​​，对于逆转录病毒生命周期早期阶段完全剪接的 HIV-1 mRNA 的产生至关重要。达 已知 SF1 与剪接体的 U2 小核核糖核蛋白亚基 (snRNP) 相关，并调节特定人类转录物的剪接。因此，Tat-SF1的研究对于理解宿主-病毒相互作用非常重要，从而为开发新的生化工具或剪接水平的治疗干预方法奠定基础。目前，Tat-SF1 在 HIV-1 和人类剪接中的潜在结构和功能作用仍不清楚。我们将通过涉及多学科方法的强有力合作来解决这一知识差距，包括前 mRNA 剪接因子的结构生物化学、分子生物学和病毒学。具体来说，我们将确定人类 U2 snRNA 和 U2 snRNP 亚基之间的 Tat-SF1 相互作用，并测试这些界面在人类剪接与 HIV-1 剪接和感染性中的功能。我们还将研究 HIV-1 抢占人类剪接机制的新作用机制，其中早期 HIV-1 Rev 蛋白通过破坏宿主 Tat-SF1-U2 snRNP 复合物来促进晚期未剪接的 HIV-1 RNA。我们已经：（i）产生了 Tat-SF1 RNA 识别基序的衍射晶体，（ii）获得了 Tat-SF1 和 U2 snRNP SF3b155 亚基之间复合物的初始结构，以及（iii）在人类细胞系中产生了 Tat-SF1 的稳定敲低。此外，我们的初步结果表明，HIV-1 Rev 蛋白与人类 Tat-SF1 相关。除了阐明 HIV-1 操纵宿主基因表达途径的新作用机制外，拟议实验的成功完成将为针对宿主辅助因子的新治疗策略奠定基础，从而规避困扰现有抗 HIV-1 治疗的耐药性挑战。