Mechanisms of vGPCR mediated Cytomegalovirus Growth in the Salivary Gland

vGPCR 介导巨细胞病毒在唾液腺中生长的机制

• 批准号: 10180884
• 负责人: WILLIAM E MILLER
• 金额: $ 40.11万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10180884
• 关键词: Adult; Affect; American; Animal Model; Antiviral Agents; Area; Biological Models; Biology; Cell model; Cell physiology; Cells; Complement; Coupled; Cytomegalovirus; Cytomegalovirus Infections; Data; Development; Disease; Ensure; Epithelial; Epithelial Cells; Fetal Development; Future; G-Protein Signaling Pathway; G-Protein-Coupled Receptors; Gene Expression; Gene Expression Profile; Genes; Genetic study; Gland; Glean; Goals; Growth; Homologous Gene; Horizontal Disease Transmission; Human; Immunocompromised Host; Implant; In Vitro; Individual; Infection; Intervention; Investigation; Knock-out; Lead; Lentivirus; Leukocytes; Life; Liquid substance; Mediating; Medical; Molecular; Movement; Murid herpesvirus 1; Mus; Neurologic; Olfactory Mucosa; Oral; Organ; Organoids; Parotid Gland; Pathogenesis; Pathologic; Pharmacology; Physiological; Physiological Adaptation; Physiological Processes; Play; Population; Positioning Attribute; Process; Property; Public Health; Publishing; Regulation; Role; Saliva; Salivary; Salivary Glands; Science; Severities; Signal Pathway; Signal Transduction; Sorting - Cell Movement; Species Specificity; Spleen; Study models; Submandibular gland; System; Testing; Tissues; Transplant Recipients; United States; Viral; Viral Pathogenesis; Viral Proteins; Virus; Virus Replication; base; cellular transduction; design; experimental study; in vivo; innovation; insight; knockout animal; mouse model; mutant; novel; prevent; salivary acinar cell; salivary cell; salivary gene; trafficking; transcriptome sequencing; transmission process; 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 that contribute to viral trafficking, persistence, and horizontal transmission is an intense and important area of investigation. Interestingly, HCMV encodes 4 genes that are homologous to cellular G-protein coupled receptors (GPCRs). The HCMV GPCRs are not essential for viral replication in vitro, however their homology to cellular GPCRs suggests that they may profoundly affect cellular physiology to ensure replication of the viruses in organs important for pathogenesis. The strict species specificity of HCMV has precluded an analysis of the function of HCMV GPCRs in vivo. However, as the biology of the related murine cytomegalovirus (MCMV) is similar to that of HCMV, the murine virus has served as a useful model for studying how the vGPCRs affect cytomegalovirus pathogenesis in vivo. Interestingly, the MCMV encoded M33 GPCR is essential for replication within the salivary gland of mice, suggesting that M33 may have a direct impact on persistence or horizontal transmission of virus. Based on our preliminary data, we hypothesize that human and murine CMV encoded vGPCRs activate similar Gaq-dependent signaling pathways to alter salivary acinar cell physiology and facilitate efficient amplification within the salivary epithelium. The proposed studies are highly significant as little is known about the viral and cellular properties that facilitate viral amplification within the gland and promote movement of virus into the saliva. In aim 1, we will use knockout and pharmacological approaches to test the hypothesis that Gaq/Ga11 is the proximal signaling pathway used by M33 for MCMV growth within the salivary gland in vivo. In aim 2, we will use novel animal models to examine cellular gene expression in salivary acinar epithelial cells infected in vivo and determine how MCMV vGPCR-induced changes provide physiological adaptations critical for optimal cytomegalovirus growth in the gland. In aim 3, we will use primary salisphere-derived organoids and test whether salivary epithelial cells similarly require CMV vGPCR activity to promote viral growth in vitro and in vivo. In aim 4 we will examine signaling properties of the HCMV and MCMV vGPCRs in the organoid systems. The innovative experiments proposed in this application will lead to important insight into the function of cytomegalovirus vGPCRs in vivo and define mechanisms by which cytomegaloviruses persist and gain access to fluids important for horizontal transmission. Defining the essential roles for cytomegalovirus vGPCRs in promoting 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）是美国的一个重大公共卫生问题。最重要的是病毒对发育中的胎儿和免疫功能低下的个体的影响，它会导致各种病理状况，严重程度从轻微到危及生命。由于 HCMV 以持久或潜伏形式存在于世界 50-90% 的成年人口中，因此鉴定有助于病毒贩运、持久性和水平传播的病毒基因产物是一个激烈而重要的研究领域。有趣的是，HCMV 编码 4 个与细胞 G 蛋白偶联受体 (GPCR) 同源的基因。 HCMV GPCR 对于病毒体外复制不是必需的，但它们与细胞 GPCR 的同源性表明它们可能深刻影响细胞生理学，以确保病毒在对发病机制重要的器官中复制。 HCMV 严格的物种特异性阻碍了对 HCMV GPCR 体内功能的分析。然而，由于相关的鼠巨细胞病毒（MCMV）的生物学特性与 HCMV 相似，因此鼠病毒已成为研究 vGPCR 如何影响巨细胞病毒体内发病机制的有用模型。有趣的是，MCMV 编码的 M33 GPCR 对于小鼠唾液腺内的复制至关重要，这表明 M33 可能对病毒的持久性或水平传播有直接影响。根据我们的初步数据，我们假设人类和小鼠 CMV 编码的 vGPCR 激活类似的 Gaq 依赖性信号通路，以改变唾液腺泡细胞生理学并促进唾液上皮内的有效扩增。拟议的研究非常重要，因为人们对促进病毒在腺体内扩增并促进病毒进入唾液的病毒和细胞特性知之甚少。在目标 1 中，我们将使用敲除和药理学方法来检验 Gaq/Ga11 是 M33 在体内唾液腺内用于 MCMV 生长的近端信号传导途径的假设。在目标 2 中，我们将使用新型动物模型来检查体内感染的唾液腺泡上皮细胞的细胞基因表达，并确定 MCMV vGPCR 诱导的变化如何提供对腺体中巨细胞病毒最佳生长至关重要的生理适应。在目标 3 中，我们将使用原代唾液球衍生的类器官，并测试唾液上皮细胞是否同样需要 CMV vGPCR 活性来促进病毒在体外和体内的生长。在目标 4 中，我们将检查类器官系统中 HCMV 和 MCMV vGPCR 的信号传导特性。本申请中提出的创新实验将有助于深入了解巨细胞病毒 vGPCR 在体内的功能，并确定巨细胞病毒持续存在并获得对水平传播至关重要的液体的机制。确定巨细胞病毒 vGPCR 在促进唾液腺复制和传播中的重要作用，最终可能导致开发出旨在防止巨细胞病毒通过唾液传播的独特抗病毒药物。