Development of salisphere-derived systems for the study of cytomegalovirus vGPCR directed viral growth in the salivary gland

开发唾液球衍生系统用于研究巨细胞病毒 vGPCR 指导唾液腺中的病毒生长

• 批准号: 9317077
• 负责人: WILLIAM E MILLER
• 金额: $ 24.53万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9317077
• 关键词: Adult; Affect; American; Animal Model; Antiviral Agents; Area; Biological Models; Biology; Cell Culture Techniques; Cell model; Cell physiology; Complement; Coupled; Cytomegalovirus; Cytomegalovirus Infections; Data; Development; Disease; Ensure; Epithelial Cells; Epithelium; Fetus; Future; G Protein-Coupled Receptor Signaling; 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; Lead; Leukocytes; Life; Liquid substance; Liver; Medical; Mental Retardation; Molecular; Movement; Murid herpesvirus 1; Mus; Neurologic; Olfactory Mucosa; Oral; Organ; Organoids; Parotid Gland; Pathogenesis; Pathologic; 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; Subfamily lentivirinae; Submandibular gland; System; Testing; Tissues; Transplant Recipients; United States; Viral; Viral Pathogenesis; Viral Proteins; Virus; Virus Replication; base; design; experimental study; humanized mouse; in vivo; innovation; insight; mouse model; novel; prevent; saliva secretion; 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 infected mice, suggesting that M33 activity may have a direct impact on viral persistence and/or horizontal transmission of virus. Based on our preliminary data, we hypothesize that human and murine CMV encoded vGPCRs activate similar Gq-dependent signaling pathways to alter salivary acinar cell physiology and facilitate efficient amplification and spread within the salivary epithelium. The proposed studies are highly significant as the salivary gland and salivary secretions play an important role in horizontal transmission of cytomegaloviruses, yet 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 primary salisphere-derived organoids and test whether implanted organoids similarly require CMV vGPCR activity to promote viral growth. In aim 2 we will examine signaling properties of the HCMV and MCMV vGPCRs in the organoid systems and in aim 3 we will use novel animal models and GFP-tagged viruses 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. 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)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%的成年人口中以持久性或潜伏性形式存在着人巨细胞病毒，因此识别有助于病毒贩运、持久性和水平传播的病毒基因产物是一个紧张而重要的调查领域。有趣的是，HCMV编码4个与细胞G蛋白偶联受体(GPCRs)同源的基因。HCMV GPCRs在体外并不是病毒复制所必需的，但它们与细胞GPCRs的同源性表明，它们可能深刻地影响细胞生理，以确保病毒在致病重要器官中的复制。由于HCMV具有严格的物种特异性，因此无法对其在体内的功能进行分析。然而，由于相关的小鼠巨细胞病毒(MCMV)的生物学特性与巨细胞病毒相似，小鼠病毒已成为研究vGPCRs如何影响巨细胞病毒体内致病的有用模型。有趣的是，MCMV编码的M33GPCR对感染小鼠唾液腺内的复制是必不可少的，这表明M33的活性可能对病毒的持久性和/或病毒的水平传播有直接影响。根据我们的初步数据，我们假设人和小鼠巨细胞病毒编码的vGPCRs激活类似的GQ依赖的信号通路来改变唾液腺泡细胞的生理，并促进唾液上皮内的有效扩增和扩散。拟议的研究具有重要意义，因为唾液腺和唾液分泌物在巨细胞病毒的水平传播中发挥着重要作用，但人们对促进病毒在腺体内放大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，我们将使用主要来自红豆杉的有机类化合物，并测试植入的有机类化合物是否同样需要CMV vGPCR活性来促进病毒生长。在目标2中，我们将研究HCMV和MCMV vGPCRs在器官系统中的信号特性，在目标3中，我们将使用新的动物模型和GFP标记的病毒来检测体内感染的唾液腺泡上皮细胞中细胞基因的表达，并确定MCMV vGPCR诱导的变化如何为巨细胞病毒在腺体中的最佳生长提供关键的生理适应。本申请中提出的创新实验将导致对巨细胞病毒vGPCRs在体内的功能的重要洞察，并确定巨细胞病毒持续存在并获得对水平传播重要的体液的机制。确定巨细胞病毒vGPCRs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独特的抗病毒药物，旨在防止巨细胞病毒通过唾液传播。