Mechanisms of vGPCR mediated Cytomegalovirus Growth in the Salivary Gland

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对小鼠唾液腺内的复制是必不可少的，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，我们假设人和小鼠CMV编码的vGPCRs激活类似的Gaq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。这项拟议的研究意义重大，因为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，我们将使用基因敲除和药理学方法来验证这样的假设，即Gaq/Ga11是M33在体内唾液腺内MCMV生长所使用的近端信号通路。在目标2中，我们将使用新的动物模型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诱导的变化如何提供对巨细胞病毒在腺体中最佳生长至关重要的生理适应。在目标3中，我们将使用原生红豆草衍生的有机化合物，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，我们将研究HCMV和MCMV vGPCRs在有机系统中的信号特性。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。

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.