Organotypic Culture as a Model for EBV Infection of Epithelial Cells

描述(申请人提供)：爱泼斯坦-巴尔病毒(EBV)与许多B淋巴细胞和上皮性恶性肿瘤有关。其中许多是艾滋病患者特有的或增加的，包括头颈部上皮癌。这项建议侧重于利用器官型培养建立EBV感染上皮细胞的体外模型。器官培养不同于单层，因为细胞分化和复层类似于原位发现的上皮。由于EBV可以在体内分化的上皮细胞中复制，器官培养是研究上皮细胞在病毒生命周期中作用的理想系统。我们的初步数据表明，我们可以有效地感染培养中的原代口腔角质形成细胞，使用表达GFP作为标记的重组EBV，我们可以产生有效模拟口腔粘膜上皮的原代口腔角质形成细胞的器官型培养。在这些初步数据的基础上，我们建议在器官培养中鉴定EBV感染的特征，鉴定感染的细胞和表达的病毒基因产物。多项研究表明，EBV可能在体内建立了一种潜伏感染。我们将确定EBV是否可以确定潜伏感染，并确定潜伏感染的类型。在上皮细胞中表达的病毒基因产物的功能将被检测。因此，我们将建立和表征一个研究上皮细胞中EBV的模型系统，并利用该系统在R21资助机制的有限范围内尽可能地确定病毒基因产物的功能。这些研究将为未来的实验奠定基础，这些实验将研究关于EBV感染人类口腔上皮的几个长期存在的问题。 公共卫生相关性：爱泼斯坦-巴尔病毒(EBV)与多种人类恶性肿瘤有关，这些恶性肿瘤主要发生在B淋巴细胞或上皮细胞，其中许多在艾滋病患者中的发病率增加。这项拟议的研究旨在建立一种EBV感染上皮细胞的体外模型，该模型将对填补我们对上皮细胞在EBV生命周期中所起作用的认识的空白具有非常重要的价值。由于口腔粘膜是病毒进入和离开的部位，上皮细胞在病毒传播中发挥着重要作用，因此，了解它们在病毒生命周期中的作用不仅有助于阐明这一重要步骤，而且有可能影响EBV感染的所有下游事件。这一模型可能不仅对增加我们对上皮细胞在病毒生命周期中的作用以及病毒对恶性肿瘤发展的贡献的知识具有重要意义，而且还可以用于开发和/或测试抗病毒策略。

DESCRIPTION (provided by applicant): Epstein-Barr virus (EBV) is associated with a number of malignancies of B lymphocyte and epithelial origin. Many of these are specific to or increased in AIDS patients including epithelial cancers of the head and neck. This proposal focuses on the development of an in vitro model of EBV infection of epithelium using organotypic cultures. Organotypic cultures differ from monolayers in that the cells differentiate and stratify to resemble epithelium found in situ. Because EBV can replicate in differentiated epithelial cells in vivo, organotypic culture is an ideal system to study the role of epithelial cells in the viral life cycle. Our preliminary data demonstrate that we can efficiently infect primary oral keratinocytes in culture using a recombinant EBV that expresses GFP as a marker, and that we can generate organotypic cultures of primary oral keratinocytes that effectively mimic oral mucosal epithelium. Based on these preliminary data, we propose to characterize EBV infection in organotypic culture identifying the cells infected and the viral gene products expressed. Several studies have suggested that EBV may establish a latent infection in vivo. We will determine whether EBV can establish a latent infection and characterize the type of latency. The functions of the viral gene products expressed in epithelium will be examined. Thus, we will establish and characterize a model system for the study of EBV in epithelium and use this system to define the functions of the viral gene products as far as possible within the limited scope of the R21 funding mechanism. These studies will set the stage for future experiments that will examine several long standing questions concerning EBV infection of human oral epithelium. PUBLIC HEALTH RELEVANCE: Epstein-Barr virus (EBV) is associated with a variety of human malignancies that occur predominantly in B lymphocytes or epithelial cells, many of which have an increased incidence in AIDS patients. The proposed research is intended to develop an in vitro model of EBV infection of epithelium that will be invaluable in filling in the gaps in our knowledge about the role of epithelial cells in the EBV lifecycle. Because the oral mucosa is the site of entry and exit of the virus, epithelial cells play a major role in viral spread and thus, understanding their role in the viral life cycle will not only shed light on this important step but has the potential to impact all downstream events from EBV infection. This model is likely to be important not only in adding to our knowledge of the role of the epithelium in the viral life cycle and the contribution of the virus to the development of malignancies, but could also be used for the development and/or testing of antiviral strategies.