Mechanisms of vGPCR mediated Cytomegalovirus Growth in the Salivary Gland

 描述(申请人提供)：人类巨细胞病毒(HCMV)在美国是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HCMV以持久性或潜伏性形式存在，因此鉴定有助于病毒贩运、持久性和水平传播的Virl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M33信号通过细胞GQ/G11途径改变唾液腺泡细胞的生理，导致病毒在唾液上皮内的扩增和传播。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特性知之甚少。在目标1中，我们将使用基因敲除、转基因和药理学方法来验证GQ/G11是M33在体内唾液腺内MCMV生长所使用的近端信号通路的假设。在目标2中，我们将测试M33改变的腺泡细胞活性或分泌物活性是否促进病毒在腺体中的扩增和/或传播。在目标3中，我们将使用新的原生唾液腺来源的“Salisphere”方法来研究唾液细胞中通过MCMV和HCMV GPCRs的信号转导，并使用这种方法来研究体内植入的唾液腺中病毒生长对HCMV GPCRs的需求。本申请中提出的创新实验将导致对巨细胞病毒GPCRs功能的新见解，并将 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GPCRs在促进唾液腺复制和传播方面的重要作用可能最终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。

 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 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 M33 signaling through the cellular Gq/G11 pathway alters salivary acinar cell physiology leading to amplification and spread of the virus 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 properties that facilitate viral amplification within the gland and promote movement of virus into the saliva. In aim 1, we will use knockout, transgenic and pharmacological approaches to test the hypothesis that Gq/G11 is the proximal signaling pathway used by M33 for MCMV growth within the salivary gland in vivo. In aim 2 we will test whether M33 altered acinar cell viability or secretor activity facilitates amplification and/or spread of virus in the gland. In aim 3 we will use novel primary salivary gland derived "salisphere" approaches to study signaling via MCMV and HCMV GPCRs in salivary cells and also use this approach to investigate the requirement for HCMV GPCRs for viral growth in salivary gland implants in vivo. The innovative 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.