Investigation of the role of HDAC activity in regulation of HCMV replication in the salivary epithelium

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和机制是一个紧张而重要的研究领域。我们最近产生并发表了一种体外模型，用于研究HCMV在称为“salispheres”的原代唾液来源的上皮细胞中的复制。这些唾液球细胞表达唾液腺泡上皮细胞的典型基因，模仿唾液腺的生理学，并作为一个有价值的模型，以了解基本参数和HCMV复制的机制在唾液腺上皮。我们令人兴奋的初步数据表明，HCMV株缺乏五聚体糖蛋白复合物可以拯救感染的唾液细胞使用组蛋白脱乙酰酶（HDAC）抑制剂。此外，我们的初步数据表明，五聚体可能需要诱导pp 71的核动员，在那里它可以与HDAC相互作用，抑制它们的活性，并促进裂解复制。我们还提出了初步的数据表明，病毒编码的GPCR（vGPCR）是必不可少的唾液细胞中的复制和药理学HDAC抑制可以类似地拯救vGPCR无效病毒所表现出的缺陷。我们的数据表明HCMV五聚体和vGPCR在促进唾液细胞裂解复制中的新作用。基于我们的初步数据，我们假设HCMV五聚体、vGPCR和pp 71协同工作以促进唾液上皮细胞中的有效病毒复制，从而导致病毒从唾液腺扩增和传播到唾液中。由于唾液腺及其分泌物在病毒的水平传播中起重要作用，因此拟议的研究对巨细胞病毒的发病机制具有重要意义，但对唾液腺上皮内促进感染和复制的病毒机制知之甚少。在目的1中，我们将测试HCMV五聚体复合物是否诱导pp 71被膜蛋白向细胞核中的移动以促进HDAC抑制。在目标2中，我们将测试HDAC 1或HDAC 3表达的敲低或pp 71的过表达是否可以拯救五聚体无效病毒以在唾液细胞中有效复制。在目标3中，我们将确定HCMV vGPCR信号传导是否与五聚体和pp 71协同工作以驱动唾液上皮细胞中的HCMV裂解复制。本申请中提出的创新实验将对参与唾液上皮细胞感染的HCMV的分子和生理特性产生重要的见解。确定唾液腺复制和传播的机制可能最终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 and mechanisms that contribute to viral trafficking, persistence, and horizontal transmission is an intense and important area of investigation. We have recently generated and published an in vitro model for studying HCMV replication in primary salivary derived epithelial cells called “salispheres”. These salisphere cells express genes typical of salivary acinar epithelial cells, mimic the physiology of the salivary gland, and serve as a valuable model to understand the basic parameters and mechanisms underlying HCMV replication in the salivary epithelium. Our exciting preliminary data indicates that HCMV strains deficient for the pentamer glycoprotein complex can be rescued for infection of salivary cells using histone deacetylase (HDAC) inhibitors. Moreover, our preliminary data suggest that the pentamer may be required to induce nuclear mobilization of pp71, where it can interact with the HDACs, inhibit their activity, and facilitate lytic replication. We also present preliminary data indicating the viral encoded GPCRs (vGPCRs) are essential for replication in salivary cells and that pharmacological HDAC inhibition can similarly rescue the defect exhibited by vGPCR null viruses. Our data suggest novel roles for both HCMV pentamer and vGPCRs in facilitating lytic replication in salivary cells. Based on our preliminary data, we hypothesize that the HCMV pentamer, vGPCRs, and pp71 work in concert to facilitate efficient viral replication in salivary epithelial cells leading to amplification and spread of virus from the salivary gland into the saliva. The proposed studies are highly significant to cytomegalovirus pathogenesis as the salivary gland and its secretions play an important role in horizontal transmission of virus, yet little is known about the viral mechanisms that facilitate infection and replication within the salivary epithelium. In aim 1, we will test whether the HCMV pentamer complex induces mobilization of the pp71 tegument protein into the nucleus to facilitate HDAC inhibition. In aim 2, we will test whether knockdown of HDAC1 or HDAC3 expression or overexpression of pp71 can rescue pentamer null viruses for efficient replication in salivary cells. In aim 3, we will determine whether HCMV vGPCR signaling works in concert with pentamer and pp71 to drive HCMV lytic replication in the salivary epithelial cells. The innovative experiments proposed in this application will generate important insight into the molecular and physiological properties of HCMV involved in salivary epithelial cell infection. Defining the mechanisms underlying salivary gland replication and spread could ultimately lead to the development of unique antivirals designed to prevent cytomegalovirus transmission via saliva.