Mechanisms of Early Growth Response Factor 1 (Egr-1) Induction by HSV-1 Lytic Inf

描述(由申请人提供)：单纯疱疹病毒1型(HSV-1)裂解性感染可引起从简单的唇疱疹到危险的角膜炎和致命性脑炎等各种疾病。许多实验室正在广泛研究病毒与宿主细胞之间的相互作用。结果表明，HSV-1在角膜细胞裂解感染过程中可快速诱导多功能转录因子早期生长反应-1(Egr-1)的表达。EGR-1是许多信号级联反应的汇聚点，在调节炎症、细胞增殖和细胞凋亡方面发挥着重要作用。Western印迹分析表明，Egr-1在Vero和兔角膜细胞系SIRC中缺失，但从感染后24小时(HPI)开始检测到Egr-1，并与用于感染的病毒数量成正比。表达EGFP的重组病毒感染后的免疫荧光研究表明，Egr1仅在感染的细胞内表达。逆转录聚合酶链式反应(RT-PCR)分析表明，由于紫外线灭活病毒启动了Egr-1的转录，但不能产生蛋白，因此Egr-1mRNA早在1HPI就已转录，不需要从头开始表达病毒基因。染色质免疫沉淀(ChIP)分析表明，HSV-1感染诱导NFkB和cAMP反应元件结合蛋白(CREB)，并在感染后募集到Egr-1启动子。综上所述，这些结果表明，Egr-1在HSV-1裂解感染后被有效诱导，并可能在病毒复制和眼部疾病进展中发挥关键作用。在本项目中，我们将进一步确定HSV-1感染诱导Egr1表达的机制，并通过以下特定目的来研究Egr1在HSV-1复制中的作用。具体目的1.了解病毒感染诱导Egr1表达的机制。在这一目标中，我们将检查是否需要细胞的病毒结合，病毒基因表达，病毒复制，或以上所有因素来启动Egr1转录/翻译。具体目的2.确定Egr1转录的调控机制。为此，我们将研究调节Egr1转录的途径。具体目的3.探讨Egr1对HSV-1基因表达和复制的影响。我们发表的数据表明，Egr1的过表达与ICP22内含子结合，并抑制ICP4和ICP22的转录。为此，我们将使用DNAzyme消除感染后Egr1的表达，并检查这种消除是否会影响HSV-1基因的表达和复制。我们的近期目标是在路易斯安那门罗大学(ULM)建立一个积极的研究项目，这样学生就可以学习病毒学，因为我们的实验室是ULM和路易斯安那州东北部唯一致力于病毒研究的研究项目。长期的任务是确定控制病毒基因表达和复制的调控机制，以开发治疗这种毁灭性和严重的病毒疾病的新的治疗方案。 公共卫生相关性：单纯疱疹病毒1型(HSV-1)的初次和反复感染可能导致角膜瘢痕形成，在美国和发达国家是导致失明的主要原因。我们发现HSV-1感染迅速诱导了角膜细胞中一种重要的多功能蛋白Egr-1的表达。该方案的完成将有助于阐明HSV-1介导的Egr1表达的分子机制，并有助于开发新的策略来预防HSV-1导致的病毒复制和失明。

DESCRIPTION (provided by applicant): Herpes simplex virus type-1 (HSV-1) lytic infection causes diseases ranging from simple cold sores to dangerous keratitis and lethal encephalitis. The interaction between virus and host cells is being investigated extensively by many laboratories. Our data demonstrated that HSV-1 can rapidly induce the expression of multi-functional transcriptional factor early growth response-1 (Egr-1) during lytic infection in corneal cells. Egr-1 functions as a convergence point for many signaling cascades and is known to play an important role in regulating inflammation, cell proliferation and apoptosis. Western blot analyses showed that Egr-1 was absent in Vero and rabbit corneal cell line SIRC but the protein was detected starting from 24 hours post infection (hpi) and was directly proportional to the amount of virus used for infection. Infection with recombinant virus expressing EGFP followed by immunofluorescent studies revealed that Egr1 was expressed only within the infected cells. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses indicated that Egr-1 mRNA was transcribed as early as 1 hpi and did not require de novo viral gene expression since UV inactivated virus initiated the Egr1 transcription but failed to produce protein. Chromatin Immunoprecipitation (ChIP) assays demonstrated that NFkB and cAMP response element binding protein (CREB) was induced by HSV-1 infection and recruited to the Egr-1 promoter upon infection. Collectively, these results suggest that Egr-1 is efficiently induced upon HSV-1 lytic infection and may play a key role in the viral replication and the disease progression in eyes. In this project, we will further identify the mechanisms that induced the Egr1 expression by HSV-1 infection and investigate the roles of Egr1 on HSV-1 replication through the following Specific Aims. Specific Aim 1. To understand how viral infection induced Egr1 expression. In this Aim, we will examine if viral binding of the cells, viral gene expression, viral replication, or all of the above were required to initiate Egr1 transcription/translation. Specific Aim 2. To determine the mechanism that controlled the transcription of Egr1. In this Aim, we will investigate the pathways that regulated Egr1 transcription. Specific Aim 3. To evaluate the effect of Egr1 on HSV-1 gene expression and replication. Our published data showed that overexpression of Egr1 bound to ICP22 intron and inhibited transcription of ICP4 and ICP22. In this aim, we will use DNAzyme to eliminate Egr1 expression by infection and check if the elimination can affect HSV-1 gene expression and replication. Our immediate goal is to establish an active research program at the University of Louisiana Monroe (ULM) so the students can study Virology since our laboratory is the only research program at ULM and Northeast Louisiana dedicating to virus research. The long-term mission is to identify the regulatory mechanisms controlling viral gene expression and replication to develop novel therapeutic protocols for treatment of this devastating and severe viral disease. PUBLIC HEALTH RELEVANCE: Herpes Simplex Virus -1 (HSV-1) primary and recurrent infection may result in scarring of the cornea and is the leading cause of blindness in US and the developed world. We showed that HSV-1 infection rapidly induced the expression of an important multifunctional protein Egr-1 in corneal cells. The completion of this proposal will shed light on the molecular mechanism of HSV-1 mediated Egr1 expression and assist to develop new strategy to prevent viral replication and blindness caused by HSV-1.