Opportunistic Oral HSV-- Mechanisms of Reactivation

机会性口腔 HSV——再激活机制

• 批准号: 6765093
• 负责人: CRAIG S MILLER
• 金额: $ 26.53万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6765093
• 关键词: DNA footprinting; HIV infections; PC12 cells; binding sites; biological signal transduction; disease /disorder model; gel mobility shift assay; gene expression; gene mutation; genetic promoter element; herpes simplex virus 1; human immunodeficiency virus; hyperthermia; immunosuppression; laboratory mouse; laboratory rabbit; latent virus infection; luciferin monooxygenase; mutant; opportunistic infections; polymerase chain reaction; protein binding; transcription factor; virus genetics; virus infection mechanism

DESCRIPTION: (Provided by Applicant) Human Immunodeficiency Virus (HIV) infection is characterized by recurrent oral disease caused by HSV-1. The defining hallmark of HSV-1 is reactivation from latency. Following stress (heat, trauma, ultra violet light, etc.) viral transcription is induced resulting in HSV-1 reactivation. The mechanism regulating the switch between latency and reactivation is poorly understood. The broad objectives of this proposal are to determine the viral gene sequences required for HSV-1 reactivation in vitro and in vivo during normal and compromised immune function. The central hypothesis to be tested is that stress induced HSV-1 genes are critical for reactivation from latency. Aim one will test the hypothesis that specific HSV-1 genes are induced by stress. Transient transfection of neurally differentiated (ND)-PC12 cells and the firefly lucifease (LUC) assay will be used to determine whether HSV-1 (i.e., alpha genes: alpha 0, alpha 4, alpha 22, alpha 27, and alpha 47, beta genes: ul9 ul23, ul39 and the gamma gene ul48 and LAT) are transcriptionally induced by heat stress and forskolin treatment in the absence of other viral gene products. Aim 2 will identify cis elements that mediate stress responsiveness of HSV-1 promoters using the LUC assay, electrophoretic mobility shift assay (EMSA) and DNA footprinting. Sequentially deleted constructs of HSV-1 promoters will be analyzed for loss of stress responsiveness in the (ND)-PCI2 cells using the LUC assay. Regions identified by standard, competition and super shift EMSA and foot printing will be assessed for stress responsiveness by mutational analysis in the LUC assay. Aim 3 will test the hypothesis that stress response elements in HSV-1 genes are required for reactivation in vitro and in vivo. The cis regulatory elements of HSV-1 genes governing the stress response will be mutated and introduced into the viral genome using recombination techniques. Mutant viruses will be tested in parallel with the rescued virus and parental virus in the PC12 cell culture model for quiescent HSV infection that was developed during their previous NIH funding. Mutant viruses that maintain wild type lytic growth properties and display at least 50 percent reduction in reactivation phenotype will be tested in the immunocompetent and immunosuppressed mouse and rabbit eye models of reactivation to determine whether altered reactivation in vitro correlates with that seen in vivo. The coordinate sequential pattern of gene expression (immediate early, early, late) of the parental, mutant and rescued viruses during the establishment and reactivation phases of quiescence will be analyzed by RT-PCR. These studies will advance the understanding of the molecular biological principals that govern the process of HSV-1 reactivation in immunocompetent and immunosuppressed individuals and could lead to the development of novel antiviral agents that block reactivation in patients with Acquired Immunodeficiency Syndrome (AIDS).

描述：（申请人提供）人类免疫缺陷病毒（HIV） 感染的特征是由HSV-1引起的复发性口腔疾病。的 定义HSV-1的标志是从潜伏期再激活。以下强制降解 (heat创伤、紫外线等）病毒转录被诱导 导致HSV-1再活化。调节两种模式之间的转换的机制 对潜伏期和再激活了解甚少。这一广泛目标 这项研究的目的是确定HSV-1所需的病毒基因序列， 在正常和受损免疫过程中的体外和体内再活化 功能待检验的中心假设是应激诱导HSV-1 基因对于从潜伏期重新激活至关重要。目标一将测试 假设特定HSV-1基因由应激诱导。瞬态 神经分化（ND）-PC 12细胞和萤火虫的转染 荧光素酶（LUC）测定将用于确定HSV-1（即，阿尔法 基因：α 0、α 4、α 22、α 27和α 47，β基因：ul 9 ul 23、ul 39和γ基因ul 48和LAT）是由 热应激和毛喉素治疗在其他病毒基因的情况下 产品.目标2将确定介导应激反应的顺式元件 使用LUC试验、电泳迁移率变动试验 （EMSA）和DNA足迹。HSV-1启动子的序列缺失构建体 将分析（ND）-PCI 2细胞中应激反应性的丧失， LUC测定。通过标准、竞争和超级转移EMSA确定的区域 和足迹将通过突变评估应激反应。 在LUC测定中进行分析。目标3将检验压力反应 HSV-1基因中的元件是体外和体内再活化所必需的。的 控制应激反应的HSV-1基因的顺式调节元件将被 突变并使用重组技术引入病毒基因组。 突变病毒将与拯救病毒和亲本病毒平行检测 在静止期HSV感染的PC 12细胞培养模型中， 在他们之前的NIH资助期间开发的。保持野生状态的变异病毒 类型裂解生长特性，并显示至少50%的减少， 再活化表型将在免疫活性和 免疫抑制小鼠和兔眼模型的再激活，以确定 体外改变的再活化是否与体内观察到的相关。的 基因表达的协调顺序模式（立即早期，早期，晚期） 在建立过程中的亲本、突变体和拯救病毒， 通过RT-PCR分析静止的再活化阶段。这些研究 将促进对分子生物学原理的理解， 控制免疫活性细胞中HSV-1再活化的过程， 免疫抑制的个体，并可能导致新的发展 抗病毒药物阻断获得性白细胞增多症患者的再激活 免疫缺陷综合症（艾滋病）。