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

人巨细胞病毒（HCMV）是美国一个重要的公共卫生问题。最重要的是病毒对发育中的胎儿和免疫功能低下的个体的影响，在这些个体中，病毒引起从轻度到危及生命的各种病理状况。由于HCMV以持久或潜伏形式存在于世界50-90%的成年人群中，因此鉴定有助于病毒运输、持久性和水平传播的病毒基因产物是一个紧张而重要的研究领域。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对于病毒体外复制不是必需的，然而它们与细胞GPCR的同源性表明它们可能深刻地影响细胞生理学以确保病毒在对发病重要的器官中复制。HCMV严格的种属特异性排除了HCMV GPCR在体内功能的分析。然而，由于相关鼠巨细胞病毒（MCMV）的生物学与HCMV相似，因此鼠病毒已成为研究vGPCR如何影响体内巨细胞病毒发病机制的有用模型。有趣的是，MCMV编码的M33 GPCR对于感染小鼠的唾液腺内的复制是必需的，这表明M33活性可能对病毒的持久性和/或水平传播具有直接影响。基于我们的初步数据，我们假设人类和小鼠CMV编码的vGPCR激活类似的GPCR依赖性信号通路，以改变唾液腺泡细胞生理学，并促进有效的扩增和唾液上皮内的扩散。由于唾液腺和唾液分泌物在巨细胞病毒的水平传播中起重要作用，因此拟议的研究非常重要，但对促进腺体内病毒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，我们将使用原代salisphere-derived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。在目标2中，我们将检查类器官系统中HCMV和MCMV vGPCR的信号传导特性，在目标3中，我们将使用新的动物模型和GFP标记的病毒来检查体内感染的唾液腺泡上皮细胞中的细胞基因表达，并确定MCMV vGPCR诱导的变化如何提供对腺体中最佳巨细胞病毒生长至关重要的生理适应。本申请中提出的创新实验将导致对巨细胞病毒vGPCR体内功能的重要见解，并定义巨细胞病毒持续存在并获得对水平传播重要的液体的机制。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，最终可能导致开发独特的抗病毒药物，以防止巨细胞病毒通过唾液传播。

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.