MECHANISTIC APPROACHES TO HSV-2 INDUCED TRANSFORMATION

单纯疱疹病毒2型（HSV-2）是一种大的DNA病毒， 它主要通过性接触传播。 宫颈癌最常发生在滥交的妇女中 或有滥交伴侣的女性。 此外，HSV-2 抗原和核酸在女性中更常见 宫颈癌的发病率要高得多。 因此，HSV-2可能是 宫颈癌的病因或几种 所需的辅助因子来体现转化状态。 的 HSV-2的BamHI-E片段诱导肿瘤形成 转化已建立的啮齿动物细胞和最小的 BamHI-E（mtrIII）的转化区映射到486个碱基对 碎片 DNA序列基序介导转化 在mtrIII中，其类似于控制重组的元件， 转录而不是病毒蛋白的表达。 的 本提案中提出的问题旨在描述 HSV-2诱导转化的机制。 做细胞原型- 癌基因在HSV-2介导的转化中起作用？ 到 回答这个问题，RNA的稳态水平， 将转化的细胞与正常细胞中的细胞进行比较。 做 mtrIII中的转录调控序列起关键作用 在HSV-2介导的转化和基因表达中的作用？ 一 细菌氯霉素乙酰转移酶报告基因 基因，将被用来关联转录增强子 转化和HSV-2表达的mtrIII活性 基因. 潜在的茎环结构和DNA构象 在mtrIII介导肿瘤转化中作用？ 位点特异 突变的茎环结构的mtrIII和影响 将分析转化时的这种突变。 的区域 改变DNA构象的mtrIII基因将被绘制出来， 使用特定的化学探针， 转型 HSV-2转化的细胞是否保留mtrIII DNA 序列？ 将分析转化细胞的整合 通过Southern印迹实验和附加体分析HSV-2序列 将鉴定含有HSV-2 DNA的元件， 在转化细胞中分析。 HSV-2是否会转化 在自然宿主人类身上的潜能 转化 将在几种人细胞系中测定HSV-2的潜力。 在 有证据表明HSV-2是导致 宫颈癌，这些研究将定义一个结构/功能 一个新的转化结构域与细胞识别的关系 在转化过程中被改变的基因。

Herpes simplex virus type 2 (HSV-2) is a large DNA virus which infects humans and is spread primarily through sexual contact. Cervical carcinoma occurs most frequently in promiscuous women or in women with promiscuous partners. Furthermore, HSV-2 antigens and nucleic acids are detected more frequently in women with cervical cancer than in those without. Thus, HSV-2 may be either the causative agent of cervical cancer or one of several cofactors required to manifest the transformed state. The BamHI-E fragment of HSV-2 induces the neoplastic transformation of established rodent cells and the minimal transforming region of BamHI-E (mtrIII) maps to a 486 base pair fragment. Transformation is mediated by DNA sequence motifs in mtrIII which resemble elements controlling recombination and transcription and not the expression of a viral protein. The questions asked in this proposal are intended to delineate the mechanism of HSV-2 induced transformation. Do cellular proto- oncogenes play a role in HSV-2 mediated transformation? To answer this question, the steady state levels of RNA in transformed cells will be compared to that in normal cells. Do the transcriptional regulatory sequences in mtrIII play a key role in HSV-2 mediated transformation and gene expression? A reporter gene, the bacterial chloramphenicol acetyltransferase gene, will be utilized to correlate the transcriptional enhancer activity of mtrIII with transformation and the expression of HSV-2 genes. Do potential stem-loop structures and DNA conformation in mtrIII mediate neoplastic transformation? Site-specific mutagenesis of a stem-loop structure in mtrIII and the effects of such mutations on transformation will be analyzed. The regions of mtrIII that have altered DNA conformation will be mapped using specific chemical probes and correlated with transformation. Do cells transformed by HSV-2 retain mtrIII DNA sequences? Transformed cells will be analyzed for integrated HSV-2 sequences by Southern blotting experiments and episomal elements which contain HSV-2 DNA will be identified and analyzed in transformed cells. Does HSV-2 posses transforming potential in its natural host, the human? The transforming potential of HSV-2 will be assayed in several human cell lines. In light of evidence which implicates HSV-2 as a causative agent of cervical cancer, these studies will define a structure/function relationship of a novel transforming domain and identify cellular genes that are altered during transformation.