Investigation of the role of HDAC activity in regulation of HCMV replication in the salivary epithelium

HDAC 活性在调节唾液上皮 HCMV 复制中的作用的研究

• 批准号: 10739852
• 负责人: WILLIAM E MILLER
• 金额: $ 24.3万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-16 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10739852
• 关键词: 3-Dimensional; Ablation; Adult; American; Antiviral Agents; Area; Biological Models; Cell Nucleus; Cell model; Cells; Clinical; Collaborations; Complement; Complex; Cytomegalovirus; Cytomegalovirus Infections; Data; Defect; Development; Disease; Epithelial Cells; Epithelium; Exhibits; Fetal Development; Future; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Genes; Glean; Glycoproteins; Goals; Growth; HDAC1 gene; HDAC3 gene; Histone Deacetylase; Histone Deacetylase Inhibitor; Horizontal Disease Transmission; Human; Immunocompromised Host; In Vitro; Individual; Infection; Intervention; Investigation; Leukocytes; Life; Liquid substance; Liver; Medical; Microcephaly; Modeling; Molecular; Movement; Neurofibromin 2; Neurologic; Nuclear; Olfactory Mucosa; Oral; Organ; Parotid Gland; Pathogenesis; Pathologic; Persons; Physiological; Physiology; Play; Population; Process; Property; Proteins; Public Health; Publishing; Regulation; Role; Saliva; Salivary; Salivary Glands; Science; Severities; Signal Transduction; Spleen; Study models; Submandibular gland; Testing; Tissues; Transplant Recipients; United States; Viral; Viral Proteins; Virus; Virus Replication; Work; design; experimental study; in vitro Model; inhibitor; innovation; insight; knock-down; lytic replication; mutant; novel; organ transplant rejection; overexpression; pharmacologic; prevent; receptor; salivary cell; trafficking; transmission process; viral rescue; viral transmission

The human cytomegalovirus (HCMV) is a significant public health concern in the United States. Most important are the effects of the virus on developing fetuses and immunocompromised individuals where it causes a variety of pathological conditions ranging in severity from mild to life-threatening. Since HCMV is present in a persistent or latent form in 50-90% of the world’s adult population, the identification of viral gene products and mechanisms that contribute to viral trafficking, persistence, and horizontal transmission is an intense and important area of investigation. We have recently generated and published an in vitro model for studying HCMV replication in primary salivary derived epithelial cells called “salispheres”. These salisphere cells express genes typical of salivary acinar epithelial cells, mimic the physiology of the salivary gland, and serve as a valuable model to understand the basic parameters and mechanisms underlying HCMV replication in the salivary epithelium. Our exciting preliminary data indicates that HCMV strains deficient for the pentamer glycoprotein complex can be rescued for infection of salivary cells using histone deacetylase (HDAC) inhibitors. Moreover, our preliminary data suggest that the pentamer may be required to induce nuclear mobilization of pp71, where it can interact with the HDACs, inhibit their activity, and facilitate lytic replication. We also present preliminary data indicating the viral encoded GPCRs (vGPCRs) are essential for replication in salivary cells and that pharmacological HDAC inhibition can similarly rescue the defect exhibited by vGPCR null viruses. Our data suggest novel roles for both HCMV pentamer and vGPCRs in facilitating lytic replication in salivary cells. Based on our preliminary data, we hypothesize that the HCMV pentamer, vGPCRs, and pp71 work in concert to facilitate efficient viral replication in salivary epithelial cells leading to amplification and spread of virus from the salivary gland into the saliva. The proposed studies are highly significant to cytomegalovirus pathogenesis as the salivary gland and its secretions play an important role in horizontal transmission of virus, yet little is known about the viral mechanisms that facilitate infection and replication within the salivary epithelium. In aim 1, we will test whether the HCMV pentamer complex induces mobilization of the pp71 tegument protein into the nucleus to facilitate HDAC inhibition. In aim 2, we will test whether knockdown of HDAC1 or HDAC3 expression or overexpression of pp71 can rescue pentamer null viruses for efficient replication in salivary cells. In aim 3, we will determine whether HCMV vGPCR signaling works in concert with pentamer and pp71 to drive HCMV lytic replication in the salivary epithelial cells. The innovative experiments proposed in this application will generate important insight into the molecular and physiological properties of HCMV involved in salivary epithelial cell infection. Defining the mechanisms underlying salivary gland replication and spread could ultimately lead to the development of unique antivirals designed to prevent cytomegalovirus transmission via saliva.

人类巨细胞病毒(HCMV)在美国是一个重大的公共卫生问题。最重要的是病毒对发育中的胎儿和免疫功能受损的人的影响，在这些人中，它会导致从轻微到危及生命的各种病理情况。由于世界上50%-90%的成年人口中存在持续或潜伏的巨细胞病毒，识别导致病毒传播、持续和水平传播的病毒基因产物和机制是一个紧张而重要的调查领域。我们最近建立并发表了一种体外模型，用于研究人巨细胞病毒在原代唾液来源的上皮细胞中的复制，该模型被称为“唾液球”。这些唾液腺细胞表达唾液腺上皮细胞的典型基因，模拟唾液腺的生理，是了解唾液上皮中HCMV复制的基本参数和机制的有价值的模型。我们令人兴奋的初步数据表明，缺乏五聚体糖蛋白复合体的HCMV毒株可以使用组蛋白脱乙酰酶(HDAC)抑制剂拯救唾液细胞感染。此外，我们的初步数据表明，五聚体可能是诱导pp71核动员所必需的，在那里它可以与HDAC相互作用，抑制它们的活性，并促进裂解复制。我们还提供了初步数据表明，病毒编码的GPCRs(VGPCRs)对于唾液细胞的复制是必不可少的，药物上的HDAC抑制同样可以修复vGPCR零病毒所表现的缺陷。我们的数据表明，HCMV五聚体和vGPCRs在促进唾液细胞裂解复制方面发挥了新的作用。根据我们的初步数据，我们假设HCMV五聚体、vGPCRs和pp71协同工作，促进唾液上皮细胞中有效的病毒复制，导致病毒从唾液腺扩增和传播到唾液中。这些研究对巨细胞病毒的发病机制具有重要意义，因为唾液腺及其分泌物在病毒的水平传播中起着重要作用，但对促进唾液上皮内感染和复制的病毒机制知之甚少。在目标1中，我们将测试HCMV五聚体复合体是否诱导pp71被膜蛋白动员到细胞核中，以促进HDAC抑制。在目标2中，我们将测试HDAC1或HDAC3表达下调或pp71过度表达是否可以拯救五聚体零病毒，以便在唾液细胞中有效复制。在目标3中，我们将确定HCMV vGPCR信号是否与五聚体和pp71协同工作，以驱动唾液上皮细胞中的HCMV裂解复制。这一应用中提出的创新实验将对HCMV参与唾液上皮细胞感染的分子和生理特性产生重要的洞察力。确定唾液腺复制和传播的机制可能最终导致开发独特的抗病毒药物，旨在防止巨细胞病毒通过唾液传播。