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

描述（由申请人提供）：1型单纯疱疹病毒（HSV-1）溶性感染导致从简单的唇疱疹到危险的角膜炎和致命的脑炎等疾病。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。我们的数据表明，HSV-1可以在角膜细胞溶解感染过程中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作为许多信号级联的汇聚点，在调节炎症、细胞增殖和细胞凋亡中发挥重要作用。Western blot分析显示，在Vero和兔角膜细胞系SIRC中不存在Egr-1，但在感染后24小时（hpi）开始检测到该蛋白，并且与用于感染的病毒量成正比。用表达EGFP的重组病毒感染后，免疫荧光研究显示Egr1仅在感染细胞内表达。逆转录聚合酶链反应（RT-PCR）分析表明，Egr-1 mRNA转录早在1 hpi，不需要从头病毒基因表达，因为紫外线灭活病毒启动了Egr1转录，但不能产生蛋白质。染色质免疫沉淀（ChIP）实验表明，NFkB和cAMP反应元件结合蛋白（CREB）在HSV-1感染时被诱导，并在感染时被募集到Egr-1启动子上。综上所述，这些结果表明Egr-1在HSV-1溶性感染中被有效诱导，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，我们将通过以下具体目的进一步确定感染HSV-1诱导Egr1表达的机制，并研究Egr1在HSV-1复制中的作用。具体目标了解病毒感染诱导Egr1表达的机制。在本研究中，我们将研究是否需要病毒结合细胞、病毒基因表达、病毒复制或上述所有条件来启动Egr1转录/翻译。具体目标2。确定控制Egr1转录的机制。在本研究中，我们将研究调控Egr1转录的途径。具体目标3。探讨Egr1对HSV-1基因表达和复制的影响。我们发表的数据显示，Egr1过表达结合ICP22内含子，抑制ICP4和ICP22的转录。为此，我们将使用DNAzyme通过感染消除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。​长期任务是确定控制病毒基因表达和复制的调节机制，以制定治疗这种破坏性和严重病毒性疾病的新治疗方案。

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.