Role of Daxx degradation by pp71 during the human cytomegalovirus life cycle

pp71 降解 Daxx 在人类巨细胞病毒生命周期中的作用

• 批准号: 8278696
• 负责人: ROBERT F KALEJTA
• 金额: $ 31.99万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-15 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8278696
• 关键词: Antiviral Agents; Antiviral Therapy; Area; Binding; Biological Models; CD34 gene; Cell Cycle; Cell Nucleus; Cells; Congenital Abnormality; Cytomegalovirus; Cytomegalovirus Infections; Cytoplasm; Data; Disease; Gene Expression; Gene Silencing; Genome; Goals; Health; Hematopoietic stem cells; Human; Immediate-Early Genes; In Vitro; Infection; Knowledge; Life Cycle Stages; Lytic; Lytic Phase; Malignant Neoplasms; Mediating; Methodology; Modeling; Modification; Molecular; Molecular Genetics; Myeloid Cells; Patients; Play; Population; Proteins; Publishing; Research Design; Resistance; Role; Signal Transduction; Testing; Time; Ubiquitin; Undifferentiated; Viral; Virus; Work; design; embryonic stem cell; immune function; immunosenescence; innovation; latent infection; lytic replication; multicatalytic endopeptidase complex; particle; pathogen; restenosis; transcription factor; transplant associated vasculopathy

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) is a significant human pathogen that infects the majority of the world's population, causes birth defects and severe disease in patients with suppressed immune function, and is associated with immunosenescence and proliferative diseases such as cancer, restenosis, and transplant-associated vasculopathy. Both primary infection and reactivations of latent infections cause HCMV disease. The HCMV pp71 protein is an attractive target for antiviral therapy because it is a transcription factor and cell cycle regulator that performs critical functions at the very beginning of both lytic and latent infections. Our long- term goal is to determine the molecular mechanisms behind each function of pp71 in HCMV-infected cells, the contribution that the different activities of pp71 make during lytic and latent viral replication cycles, and to use this knowledge to design antiviral strategies against HCMV that target the pp71 protein. pp71 is a tegument protein that is delivered to cells by infectious particles and controls the expression of the viral immediate early (IE) genes. IE gene expression initiates lytic replication, and must be silenced in order for the virus to establish latency. Our published work shows that a cellular protein called Daxx silences HCMV IE gene expression, and that during a lytic infection, tegument-delivered pp71 localizes to the nucleus and degrades Daxx in a proteasome-dependent, ubiquitin-independent manner to stimulate IE gene expression (63,132). Our new preliminary data indicates that pp71 is necessary and sufficient to induce a modified form of Daxx. Because the timing of this modification correlates with Daxx degradation, we will test if this modification of Daxx is the ubiquitin-independent signal for Daxx degradation. We have also recently published that tegument-delivered pp71 is trapped in the cytoplasm and thus fails to degrade Daxx in cells where HCMV establishes a quiescent or latent-like infection in vitro (133). In these cells, IE genes are silenced in a Daxx-dependent manner. Thus, Daxx stability upon HCMV infection determines whether infected cells initiate lytic, or establish latent- like infections. Our new preliminary evidence indicates that Daxx and pp71 also control true latent infections established in CD34+ undifferentiated myeloid cells. Because CD34+ cells are difficult to maintain as undifferentiated cells in culture, we are developing embryonic stem cells as a more versatile model to study HCMV latency. Here we propose to further our examination of the role that pp71-mediated degradation of Daxx plays during HCMV latency, and to define the molecular mechanism through which pp71 degrades Daxx. This proposal builds upon our published and unpublished discoveries, utilizes state of the art molecular and genetic approaches, and introduces creative and innovative new methodologies to study HCMV latency. PUBLIC HEALTH RELEVANCE: Almost everyone is infected with human cytomegalovirus (HCMV), and once infected, can never get rid of the virus. HCMV causes disease in young, old, sick, and otherwise healthy patients. We are studying how the virus begins to replicate in the hope of finding ways to stop viral replication and thus treat HCMV disease.

描述(申请人提供)：人类巨细胞病毒(HCMV)是一种重要的人类病原体，感染世界上大多数人口，导致出生缺陷和免疫功能受抑患者的严重疾病，并与免疫衰老和增殖性疾病有关，如癌症、再狭窄和移植相关血管病变。初次感染和潜伏感染的重新激活都会导致巨细胞病毒病。HCMV pp71蛋白是一种转录因子和细胞周期调节因子，在裂解性感染和潜伏性感染的初期发挥关键作用，因此是抗病毒治疗的一个有吸引力的靶点。我们的长期目标是确定在HCMV感染细胞中pp71每种功能背后的分子机制，以及pp71的不同活性在裂解和潜伏病毒复制周期中的贡献，并利用这一知识设计针对pp71蛋白的抗HCMV策略。PP71是一种被膜蛋白，通过感染颗粒传递到细胞，控制病毒即刻早期(IE)基因的表达。IE基因表达启动裂解复制，必须沉默才能使病毒建立潜伏期。我们发表的工作表明，一种名为Daxx的细胞蛋白抑制HCMV IE基因的表达，在裂解感染期间，被膜递送的pp71定位于细胞核，并以蛋白酶体依赖、泛素不依赖的方式降解Daxx，以刺激IE基因的表达(63,132)。我们新的初步数据表明，pp71是诱导Daxx修饰形式所必需的，也是充分的。由于这种修饰的时机与Daxx的降解有关，我们将测试Daxx的这种修饰是否是Daxx降解的泛素独立信号。我们最近还发表了被膜传递的pp71被困在细胞质中，因此不能在细胞中降解Daxx，而在体外HCMV建立了静止或潜伏的感染(133)。在这些细胞中，IE基因以依赖于Daxx的方式沉默。因此，Daxx对HCMV感染的稳定性决定了感染细胞是启动裂解，还是建立潜伏性感染。我们的新的初步证据表明，Daxx和pp71也控制了CD34+未分化髓系细胞中建立的真正的潜伏感染。由于CD34+细胞在培养中很难维持为未分化细胞，我们正在开发胚胎干细胞作为一种更通用的模型来研究HCMV潜伏期。在这里，我们建议进一步研究pp71介导的Daxx降解在HCMV潜伏期中所起的作用，并确定pp71降解Daxx的分子机制。这项建议建立在我们已发表和未发表的发现的基础上，利用最先进的分子和遗传学方法，并引入创造性和创新性的新方法来研究HCMV潜伏期。公共卫生相关性：几乎每个人都感染了人类巨细胞病毒(HCMV)，一旦感染，永远无法摆脱该病毒。人巨细胞病毒在年轻、年老、生病和其他健康的患者中引起疾病。我们正在研究病毒如何开始复制，希望找到阻止病毒复制从而治疗HCMV疾病的方法。