Opportunistic Oral HSV-- Mechanisms of Reactivation

机会性口腔 HSV——再激活机制

• 批准号: 6516664
• 负责人: CRAIG S MILLER
• 金额: $ 26.69万
• 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/6516664
• 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。这个 定义HSV-1的特征是从潜伏期重新激活。追随压力 (高温、创伤、紫外线等)病毒转录被诱导 导致HSV-1重新激活。调节两种基因之间的转换的机制 人们对潜伏期和重新激活知之甚少。这个项目的总体目标是 建议确定HSV-1所需的病毒基因序列 免疫正常和免疫受损时的体外和体内再激活 功能。需要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。目标一号将测试 假设特定的HSV-1基因是由应激诱导的。瞬变 神经分化(ND)-PC12细胞和萤火虫的转基因 荧光素酶(Luc)试验将用于确定HSV-1(即阿尔法)是否 基因：阿尔法0，阿尔法4，阿尔法22，阿尔法27，阿尔法47，贝塔基因：ul9 Ul23、ul39和伽马基因ul48和lat)是由 热应激和无其他病毒基因的Forsklin治疗 产品。目标2将确定调节应激反应的顺式元件。 用LUC法、凝胶迁移率改变法检测HSV-1启动子 (EMSA)和DNA足迹。HSV-1启动子序列缺失的构建 将分析(ND)-PCI2细胞中的应激反应丧失 LUC化验。由标准、竞争和超级班次EMSA确定的地区 脚印将通过突变来评估应激反应 在LUC检测中的分析。目标3将检验压力反应的假设 HSV-1基因中的元件是在体外和体内重新激活所必需的。这个 控制应激反应的HSV-1基因的顺式调控元件将是 利用重组技术突变并引入病毒基因组。 变异病毒将与被拯救的病毒和亲本病毒同时进行测试 病毒在PC12细胞培养模型中的静止期HSV感染 是在他们之前的NIH资助期间开发的。保持野性的突变病毒 键入裂解生长特性，并显示至少50%的减少 重新激活表型将在免疫活性和 免疫抑制小鼠和兔眼模型的再激活测定 体外改变的再激活是否与体内看到的相关。这个 基因表达的协调顺序模式(即刻早、早、晚) 亲本病毒、突变病毒和被拯救的病毒在建立和 静止期的复活阶段将通过RT-PCR进行分析。这些研究 将促进对分子生物学原理的理解 控制HSV-1在免疫和免疫系统中的重新激活过程 免疫抑制的个体，并可能导致新的发展 抗病毒药物可阻断获得性心力衰竭患者的再激活 免疫缺陷综合症(艾滋病)。