Innate Corneal Cell Immunity to Virus Infection

角膜细胞对病毒感染的先天免疫力

基本信息

批准号：
6776346
负责人：
Jerry L Taylor
金额：
$ 22.5万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-08-01 至 2006-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6776346
关键词：
antigen antibody reaction binding proteins cellular immunity cornea fluorescence microscopy gene expression gene induction /repression genetic transcription green fluorescent proteins herpes simplex virus 1 human tissue interferons ocular herpes phosphorylation protein isoforms virus infection mechanism yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Infection of the cornea with herpes simplex virus (HSV) initiates an innate immune response that plays a major role in limiting virus spread. Production of interferon (IFN) and IFN-induced proteins is an important component of this innate response. Some of the IFN-inducible proteins localize to nuclear structures termed ND1Os, which are the sites of viral DNA localization and initiation of viral transcription. The aims of this grant focus on determining the mechanism of action of two of these IFN-induced proteins, PML and SP100, and the regulation of their expression in human cornea (HCS) cells. Each protein exists in multiple forms as a result of variable mRNA splicing. Our studies show that SP100B is a potent inhibitor of two HSV transcriptional transactivating proteins, VP16 and ICP4. The specificity of SP100B's inhibitory activity will be characterized by examining the action against basal expression and transactivation of a number of viral and cellular promoters. Using the yeast two-hybrid system we will identify cellular protein(s) that function as mediators between SP100B and ICP4. The region of SP100B that confers repression is within a 29 amino acid domain that has the potential to be highly phosphorylated. This region will be mapped to determine whether specific amino acid phosphorylation is essential for activity. We will determine whether localization to the ND1O structure and transcriptional repressive activity are linked. The second protein to be examined is PML, a structural component of ND1Os. Two forms of PML will be evaluated alone and combined with SP100B for their contribution to the transcriptional regulation of HSV genes. The interaction of PML with the viral general transcriptional transactivator ICPO, a protein known to disrupt ND1s, will be characterized by transcription assays in transiently and stably transfected cells and by microscopic localization of green fluorescent protein-tagged forms of PML. HCS cells appear to contain high levels of PML localized to ND1Os, potentially acting to provide an elevated innate immune response. Monoclonal antibodies specific for forms of PML and SP100 will be prepared and used to identify the forms of PML and SP100 naturally present in these corneal cells and determine the changes that occur in their expression in response to IFN treatment and virus infection. These studies will characterize a potent transcriptional repressive mechanism we have identified and determine its contribution to corneal inhibition of HSV replication. This knowledge may be utilized to enhance this innate antiviral response and thereby limit initial and recurrent viral infections in the eye.

描述（由申请人提供）：带有疱疹的角膜感染单纯形病毒（HSV）启动了先天免疫反应，起着主要作用在限制病毒扩散中。干扰素（IFN）和IFN诱导的产生蛋白质是这种先天反应的重要组成部分。一些 IFN诱导蛋白本地化为称为ND1OS的核结构，病毒DNA定位的位点和病毒转录的启动。这这笔赠款的目的重点是确定其中两个的作用机理 IFN诱导的蛋白质，PML和SP100，并调节其表达人角膜（HCS）细胞。每种蛋白质以多种形式存在于可变的mRNA剪接。我们的研究表明，SP100B是两个HSV转录反式激活蛋白VP16和ICP4。这 SP100B抑制活性的特异性将通过检查来表征反对基础表达和许多病毒的反式激活的作用和细胞启动子。使用酵母双杂交系统，我们将确定在SP100B和ICP4之间充当介体的细胞蛋白。这赋予抑制的Sp100b区域在29个氨基酸结构域内具有高度磷酸化的潜力。该区域将被映射到确定特定的氨基酸磷酸化是否对于活动。我们将确定定位到ND1O结构和转录抑制活性相关。第二种蛋白质是检查的是ND1OS的结构成分PML。两种形式的PML将是单独评估并与SP100B合并，以贡献 HSV基因的转录调节。 PML与病毒的相互作用一般转录反式激活器ICPO，一种已知破坏ND1S的蛋白质，将以瞬时和稳定的转录测定为特征转染的细胞和通过微观定位绿色荧光蛋白质标记的PML形式。 HCS细胞似乎包含高水平的PML 局部到ND1OS，有可能采取行动以提供升高的先天免疫回复。针对PML和SP100形式的单克隆抗体将是准备并用于识别自然存在的PML和SP100的形式这些角膜细胞并确定其表达中发生的变化对IFN治疗和病毒感染的反应。这些研究将表征我们已经确定并确定的有效的转录抑制机制它对角膜抑制HSV复制的贡献。这些知识可能被用来增强这种先天的抗病毒反应，从而限制了初始眼睛中的病毒感染。