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.

在美国，人类巨细胞病毒（HCMV）是一个重大的公共卫生问题。最重要的是该病毒对发育中的胎儿和免疫功能低下的个体的影响，在这种情况下会导致各种病理状况，从而严重程度从轻度到威胁生命。由于HCMV以持续或潜在的形式存在于全球成年人口的50-90％中，因此鉴定了导致病毒贩运，持久性和水平传播的病毒基因产品的鉴定是一个强烈而重要的投资领域。有趣的是，HCMV编码与细胞G蛋白偶联受体（GPCR）同源的4个基因。 HCMV GPCR对于体外病毒复制并不是必不可少的，但是它们与细胞GPCR的同源性表明，它们可能会深刻影响细胞生理学，以确保器官中病毒的复制对发病机理重要。 HCMV的严格规格特异性排除了对体内HCMV GPCR功能的分析。然而，由于相关鼠巨细胞病毒（MCMV）的生物学类似于HCMV，因此鼠病毒已成为研究VGPCR如何影响巨细胞病毒发病机理的有用模型。有趣的是，MCMV编码的M33 GPCR对于在感染小鼠的唾液腺内复制至关重要，这表明M33活性可能对病毒持久性和/或水平传播具有直接影响。根据我们的初步数据，我们假设人和鼠CMV编码VGPCR会激活类似的GQ依赖性信号传导途径，以改变唾液腺泡细胞生理学，并促进有效的扩增并在唾液上皮中扩散。拟议的研究非常重要，因为唾液腺和唾液分泌物在巨细胞病毒的水平传播中起着重要作用，但对促进腺体内病毒扩增并促进病毒运动并促进病毒运动的病毒和细胞特性知之甚少。在AIM 1中，我们将使用原发性硅球来源的类器官，并测试植入的类器官是否同样需要CMV VGPCR活性来促进病毒生长。在AIM 2中，我们将检查器官系统中HCMV和MCMV VGPCR的信号传导特性，在AIM 3中，我们将使用新颖的动物模型和GFP标记的病毒来检查唾液腺泡上皮细胞中感染体内感染的细胞基因的表达，并确定MCMV VGPCR诱导的变化对物理适应的变化，以提供敏捷的适应性。本应用中提出的创新实验将导致对体内巨细胞病毒VGPCR的功能的重要见解，并定义机制，通过该机制，巨细胞病毒持续存在并获得对水平传播重要的流体。定义巨细胞病毒VGPCR在促进唾液腺复制和扩散中的基本作用最终可能导致开发旨在防止通过唾液传播的独特抗病毒毒药。