SIGNAL TRANSDUCTION IN ORAL HERPES REACTIVATION

调节潜伏性单纯疱疹病毒再激活的机制 暴露于外界刺激后的1型(HSV-1)仍是一个谜 尽管进行了多年的研究。目前的知识状况认识到 大约0.1%的潜伏感染感觉神经节神经元 含有HSV-1 DNA，潜伏的病毒基因组维持在神经元中 低拷贝数的细胞核，HSV-1基因组的表达 在延迟期间受到极大限制。潜伏病毒的唯一区域 基因组转录活性是在长重复序列中发现的一种基因 地区。一个被称为潜伏期相关转录本(LAT)的RNA家族 都是由这种二倍体基因转录的。而LAT的功能不是 然而，最近明确的证据表明，LAT可以限制HSV-1 重新激活，并且各种刺激通过信号诱导重新激活 释放其抑制作用的转导。这一假设是 我们的发现支持：i)来源于神经细胞的蛋白质 与LAT启动子的一个区域(-280至-238)特异性结合 暴露在紫外线(UV)光和热休克(HS)下，II)紫外线 响应要素(AP1)存在于该区域内，以及iii) 紫外线照射下神经细胞LATs的表达下调 瞬时氯霉素乙酰转移酶测定辐射 (CAT)测定。 该项目将进一步探索DNA结合蛋白的作用。 紫外线和HS诱导LATs的表达及信号的作用 口服HSV-1激活过程中的转导。LAT的启动子序列 (HSV-1)与紫外线和HS诱导的蛋白结合的病毒将使用 电泳迁移率改变分析和DNase I足迹。至 确定这些蛋白质是否是转录因子、暂时性CAT 检测将使用携带5‘缺失的报告质粒进行 拉特斯。小鼠lambdagt11基因表达文库的构建 将对神经母细胞瘤细胞进行筛选，以确定编码 LATS结合蛋白。将对该基因的序列进行分析以 确定它编码的是已知的蛋白质还是新的蛋白质。该基因将被命名为 用于共转染实验，以确定表达的蛋白是否 调节LATS的表达。从cDNA克隆产生的探针 将被用来研究潜伏的小鼠的重新激活 感染HSV-1野生型或HS结合位点缺失的HSV-1(S)， 在体温过高后。单纯疱疹病毒1型病毒再激活的特征 将通过外植体培养潜伏感染的神经节和 通过原位杂交。这些研究应该能让我们深入了解 外界刺激诱导HSV-1重新激活的机制。

The mechanisms that regulate reactivation of latent herpes simplex virus type 1 (HSV-1) after exposure to external stimuli remain an enigma despite years of research. The current state of knowledge recognizes that approximately 0.1% of latently infected neurons of sensory ganglia contain HSV-1 DNA, the latent viral genome is maintained in neuronal nuclei in low copy number, and the expression of the HSV-1 genome is extremely restricted during latency. The only region of the latent viral genome transcriptionally active is a gene found within the long repeat regions. A family of RNAs termed latency associated transcripts (LATs) are transcribed by this diploid gene. While the function of LATs is not yet clear, recent evidence has suggested that LATs restrict HSV-1 reactivation, and various stimuli induce reactivation via signal transduction that release its repressive effects. This hypothesis is supported by our findings that i) proteins derived from neuronal cells bind specifically to a region (-280 to -238) of the LAT promoter after exposure to ultraviolet (Uv) light and heat shock (HS), ii) an UV response element (AP1) is present within this region, and iii) the expression of LATs is down-regulated in neuronal cells exposed to UV irradiation as determined by transient chloramphenicol acetyltransferase (CAT) assay. This project will further explore the role of DNA binding proteins induced by UV and HS in the expression of LATs, and the role of signal transduction in oral HSV-1 reactivation. The promoter sequences of LATs (HSV-1) that bind proteins induced by UV and HS will be identified using electrophoretic mobility shift assay and DNase I footprinting. To determine if these proteins are transcription factors, transient CAT assays will be performed using reporter plasmids carrying 5' deletions of LATs. A lambdagt11 cDNA expression library made from mouse neuroblastoma cells will be screened to identify the gen encoding the LATs binding protein. The sequence of the cDNA will be analyzed to determine if it encodes a known or a novel protein. The cDNA will be used in co-transfection experiments to determine if the expressed protein regulates the expression of LATS. Probes generated from the cDNA clone will be used to study reactivation in mice that have been latently infected with HSV-1 wild-type or HSV-1 deleted of the HS binding site(s), after exposure to hyperthermia. Characterization of HSV-1 reactivation will be performed by explant cultivation of latently infected ganglia and by in situ hybridization. These studies should provide insight into the mechanism by which external stimuli induce HSV-1 reactivation.