Role of Cytomegalovirus GPCRs in Pathogenesis in Vivo

描述（由申请人提供）：人巨细胞病毒（HCMV）是美国的一个重要公共卫生问题。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以持续或潜伏形式存在于世界上50-90%的成年人群中，因此鉴定有助于病毒运输、持续和水平传播的病毒1基因产物是一个紧张而重要的研究领域。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，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体内巨细胞病毒复制和运输方面的有用模型。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M33通过细胞Gaq/Ga 11途径的信号传导改变了唾液腺泡细胞的生理学，导致病毒在唾液腺内的扩增和传播。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特性知之甚少。在目标1中，我们将检验M33无效病毒由于腺泡上皮内病毒传播缺陷而不能在唾液腺中扩增的假设。在目标2中，我们将测试M33是否改变腺泡细胞活力或分泌活性，促进病毒在腺体中的扩增和/或传播。在上午3，我们将使用病毒和小鼠敲除方法来测试的假设，Gaq/Ga 11是近端信号通路所使用的M33的MCMV运输和生长在唾液腺在体内。我们将利用M33基因敲除和野生型病毒，敲除和转基因动物，以及唾液腺功能的生理学分析来探索我们的假设。本申请中提出的创新体内实验将导致对巨细胞病毒GPCR的功能以及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的新见解。确定巨细胞病毒GPCR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。

DESCRIPTION (provided by applicant): 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 virl 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 aspects of cytomegalovirus replication and trafficking 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 M33 signaling through the cellular Gaq/Ga11 pathway alters salivary acinar cell physiology leading to amplification and spread of the virus within the salivary gland. 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 properties that facilitate viral amplification within the gland and promote movement of virus into the saliva. In aim 1 we will test the hypothesis that M33 null viruses fail to amplify in the salivary gland due to defectie viral spread within the acinar epithelium. In aim 2 we will test whether M33 altered acinar cell viability or secretory activity facilitates amplification and/or spread of virus in the gland. In am 3 we will use viral and murine knockout approaches to test the hypothesis that Gaq/Ga11 is the proximal signaling pathway used by M33 for MCMV trafficking and growth in the salivary gland in vivo. We will utilize M33 knockout and wildtype viruses, knock- out and transgenic animals, and physiological analyses of salivary gland function to explore our hypothesis. The innovative in vivo experiments proposed in this application will lead to novel insights into the function of cytomegalovirus GPCRs and into mechanisms by which cytomegaloviruses persist and gain access to fluids important for horizontal transmission. Defining essential roles for cytomegalovirus GPCRs in promoting salivary gland replication and spread could ultimately lead to the development of unique antivirals designed to prevent cytomegalovirus transmission via saliva.