MECHANISTIC APPROACHES TO HCMV MTRII AND MTRIII-INDUCED TRANSFORMATION

人类巨细胞病毒与多种人类疾病有关 恶性疾病，如神经母细胞瘤、前列腺癌、宫颈癌 癌症和结肠癌。 该病毒在艾滋病和艾滋病中重新激活 移植患者。 其转型潜力备受关注 在疫苗研发方面。我们报告了长链中的两个转化域 人类巨细胞病毒 (HCMV) 基因组的独特片段：i) mtrII (980 bp) ii) mtrIII (7.5 kb)。 研究了变换函数 产生缺失克隆，通过DNA介导的基因转染克隆 转移至小鼠 3T3 或 Rat-2 细胞系并分析细胞 琼脂糖中的贴壁独立生长和小鼠的致瘤性。 发现 mtrII 区域包含 3 个开放阅读框 (ORF) (79, 83 和 34 aa) 通过 DNA 序列分析。我们还报道了发起人 使用 CAT 分析检测 mtrII 中 ORF 上游区域的活性 转化子中 mtrII 转录物的检测。相似的mRNA 在 HCMV 感染的细胞中也检测到了这些物种。沿着这些思路， mtrII 转化活性也在人体细胞中进行了测试。我们最近 确定 mtrII 能够转化人类表皮细胞 角质形成细胞系（RHEK-1）具有致瘤性，并且肿瘤是 诊断为低分化癌。 进一步的实验将 有必要通过以下方式确定ORF在转化中的作用 进一步缺失克隆的产生或 ORF 的诱变。还有 将尝试对转录本进行映射以确定是否 相同的基因在转化细胞和感染细胞中表达。 关于 mtrIII (7.5 kb) 转化域，CMV 主要直接 其中包含早期基因（IE-1）。该 IE-1 基因是候选基因 亚单位疫苗。我们的删除分析定位了转化 mtrIII 对 IE-1 基因之外 2.1 kb 区域的活性。该地区还 包含多个 ORF，目前正在对其进行调查 转型中的作用。 这些研究将适用于产生安全的活减毒 CMV 疫苗，亚单位疫苗，最重要的是使用 CMV 作为克隆 基因治疗的载体。

Human cytomegalovirus has been linked to numerous types of human malignant diseases such as neuroblastoma, prostate cancer, cervical cancer and colon carcinoma. The virus is reactivated in AIDS and in transplant patients. Its transforming potential is of great concern in vaccine development. We reported two transforming domains in the long unique segment of human cytomegalovirus (HCMV) genome: i) mtrII (980 bp) and ii) mtrIII (7.5 kb). Transforming functions were studied by generating deletion clones, transfecting the clones by DNA mediated gene transfer into mouse 3T3 or Rat-2 cell lines and assaying the cells for anchorage independent growth in agarose and for tumorigenicity in mice. The mtrII region was found to contain 3 open reading frames (ORFs) (79, 83 and 34 aa) by DNA sequence analysis. We also reported the promoter activity in the upstream region of ORFs in mtrII using CAT assays and the detection of mtrII transcripts in the transformants. Similar mRNA species were also detected in HCMV infected cells. Along these lines, mtrII transforming activity was also tested in human cells. We recently identified that mtrII was capable of transforming human epidermal keratinocyte cell line (RHEK-1) to tumorigenicity, and the tumors were diagnosed as poorly differentiated carcinoma. Further experiments will be necessary to identify the role of the ORFs in transformation by generation of further deletion clones or mutagenesis of the ORFs. Also mapping of the transcripts will be attempted to identify whether the same gene is expressed in both the transformed and the infected cells. Regarding mtrIII (7.5 kb) transforming domain, the CMV major immediate early gene (IE-1) is contained within it. This IE-1 gene is a candidate subunit vaccine. Our deletion analysis localized the transforming activity of mtrIII to a 2.1 kb region beyond IE-1 gene. This region also contained several ORFs which are currently under investigation for their role in transformation. These studies will be applicable to generate a safe live attenuated CMV vaccine, a subunit vaccine and most importantly to use CMV as a cloning vehicle for gene therapy.