Epigenetic control of HCMV latency and reactivation

HCMV 潜伏期和再激活的表观遗传控制

• 批准号: 8934956
• 负责人: MARY A HUMMEL
• 金额: $ 51.44万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8934956
• 关键词: Acute; Antiviral Agents; Binding; Binding Sites; CD34 gene; Cell Lineage; Cell Nucleus; Cells; ChIP-seq; Characteristics; Chromatin; Complex; Cytomegalovirus; DNA Binding; Dendritic Cells; Early Promoters; Environment; Enzymes; Epigenetic Process; Gene Expression; Genes; Genetic Transcription; Genome; Hematopoietic; Hematopoietic stem cells; Histone Deacetylase; Histones; Immediate-Early Genes; Immediate-Early Proteins; Infection; Inflammation; Inflammation Mediators; Inflammatory; Interleukin-6; Latent Virus; Lipopolysaccharides; Lytic; Lytic Phase; Mediating; Methylation; Modeling; Modification; Morbidity - disease rate; Myelogenous; Myeloid Progenitor Cells; Phenotype; Play; Process; Proteomics; Repression; Role; Therapeutic Intervention; Transcription Regulatory Protein; Transplant Recipients; Transplantation; Tumor Necrosis Factor-alpha; Viral; Viral Genes; Viral Genome; Virion; Virus; activating transcription factor; allograft rejection; chromatin remodeling; cytokine; epigenome; graft vs host disease; histone modification; inhibitor/antagonist; insight; lytic replication; monocyte; mortality; mutant; novel strategies; pathogen; prevent; progenitor; programs; reactivation from latency; transcription factor; viral DNA

ABSTRACT. Reactivation of latent human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality in transplant recipients, despite the use of antiviral drugs. Therefore, new approaches are required to reduce the complications from this pathogen. Previous studies have shown that HCMV establishes latency in myeloid lineage cells, including CD34+ hematopoietic progenitor cells (HPCs). These pluripotent cells have a unique epigenetic environment in which HDAC expression is low, and many genes are transcriptionally inactive, but carry bivalent chromatin marks (repressive H3K27me3 and activating H3K4me3) characteristic of facultative chromatin. We hypothesize that HCMV exploits the unique epigenetic environment of HSCs, so that most lytic genes are repressed through bivalent histone modification, but the viral genome is poised to reactivate under appropriate stimulation. The major immediate early (IE) genes encode transcriptional regulatory proteins required to activate lytic infection. Expression of these genes is controlled by the major immediate early promoter (MIEP), which carries binding sites for both activating and repressive transcription factors. We further hypothesize that repressive cellular transcription factors bind to the MIEP to mediate heterochromatinization of the genome in HPCs, and that HCMV reactivation requires 1) a switch in factors binding to the MIEP, from repressive to activating transcription factors; 2) recruitment of co-activator complexes through interaction with DNA-binding partners; 3) reprogramming of viral chromatin by enzymes that mediate histone modifications. We will investigate these hypotheses using experimentally infected CD34+ cells as a model for latency and differentiation to a dendritic cell phenotype as a model for reactivation. In Aim 1 we will 1) analyze modification of histones bound to viral genomes in latently infected hematopoietic progenitor CD34+ cells and in reactivated CD34-derived DCs; 2) correlate differentiation- dependent changes in the epigenome with changes in gene expression; and 3) investigate the requirement for H3K9 de-methylation in lytic infection of permissive cells and in reactivation in CD34-derived DCs. In Aim 2 we will investigate the role of transcription factors that bind to the MIEP in latency and reactivation. De- repression of early gene expression is a second key step in both lytic infection and in reactivation from latency. In Aim 3 we use a mutant virus that conditionally expresses IE proteins to investigate the role of the IE proteins in reprogramming viral chromatin to activate lytic replication. Through the use of state-of-the art epigenetics, proteomics, and functional analyses, our studies will have determined mechanisms that regulate viral chromatin in latency and reactivation, and identified potential new targets for therapeutic intervention to prevent reactivation of CMV. This project synergizes with Projects 1 and 3, which will investigate the roles of inflammation and epigenetics in control of MCMV in latency and reactivation.

摘要。潜伏的人巨细胞病毒（HCMV）的再激活仍然是发病的重要原因 和移植受者的死亡率，尽管使用了抗病毒药物。因此，新的方法是 以减少这种病原体引起的并发症以前的研究表明，HCMV 在骨髓谱系细胞中建立潜伏期，包括CD 34+造血祖细胞（HPC）。这些 多能细胞具有独特的表观遗传环境，其中HDAC表达低，并且许多基因 转录失活，但携带二价染色质标记（抑制性H3 K27 me 3和激活性H3 K27 me 3）。 H3 K4 me 3）兼性染色质的特征。我们假设HCMV利用了独特的表观遗传学机制， 因此，大多数裂解基因通过二价组蛋白修饰而被抑制，但 病毒基因组在适当的刺激下会重新激活。立即早期（immediate early，IE）基因 编码激活裂解性感染所需的转录调节蛋白。这些基因的表达是 由主要立即早期启动子（MIEP）控制，MIEP携带激活和激活的结合位点， 抑制性转录因子。我们进一步假设，抑制性细胞转录因子与 MIEP介导HPC中基因组的异染色质化，HCMV再活化需要1） 与MIEP结合的因子从抑制性转录因子转变为激活性转录因子; 2） 通过与DNA结合伴侣相互作用的共激活因子复合物; 3）病毒染色质的重编程 通过酶介导组蛋白修饰。我们将通过实验来研究这些假设。 感染的CD 34+细胞作为潜伏期和分化为树突状细胞表型的模型， 重新激活在目标1中，我们将1）分析在潜伏感染中与病毒基因组结合的组蛋白的修饰， 造血祖细胞CD 34+细胞和再活化的CD 34-衍生的DC中; 2）相关分化- 表观基因组的依赖性变化与基因表达的变化;和3）调查的要求 对于H3 K9去甲基化在允许细胞的裂解性感染中和在CD 34衍生的DC中的再活化。在Aim中 2我们将研究与MIEP结合的转录因子在潜伏期和重新激活中的作用。去- 早期基因表达的抑制是溶解性感染和从感染中重新激活的第二个关键步骤。 延迟。在目的3中，我们使用条件性表达IE蛋白的突变病毒来研究IE蛋白的作用。 IE蛋白在重编程病毒染色质以激活裂解复制中的作用。通过使用最先进的 表观遗传学、蛋白质组学和功能分析，我们的研究将确定调节 病毒染色质的潜伏期和再激活，并确定了潜在的新的治疗干预目标， 防止CMV再激活。该项目与项目1和项目3协同作用，项目1和项目3将调查以下方面的作用： 炎症和表观遗传学控制MCMV潜伏和再激活。