MECHANISTIC APPROACHES TO HCMV MTRII AND MTRIII-INDUCED TRANSFORMATION

人类巨细胞病毒与许多类型的人类 恶性疾病，如神经母细胞瘤、前列腺癌、宫颈癌 癌症和结肠癌。 这种病毒在艾滋病中被重新激活， 移植患者 它的转化潜力令人非常关注 在疫苗研发中。我们报道了两个转化结构域在长 人巨细胞病毒（HCMV）基因组的独特片段：i）mtrII（980 bp） 和ii）mtrIII（7.5kb）。 通过以下方法研究了转换函数： 产生缺失克隆，用DNA介导的基因转染法对克隆进行测序， 转移到小鼠3 T3或Rat-2细胞系中，并测定细胞的 琼脂糖中的锚定非依赖性生长和小鼠中的致瘤性。 发现mtrII区含有3个开放阅读框（ORF）（79， 83和34 aa）。我们还报道了启动子 使用CAT测定在mtrII中ORF上游区域的活性， 在转化体中检测mtrII转录物。相似mRNA 在HCMV感染的细胞中也检测到两种病毒。沿着这些路线， 还在人细胞中测试了mtrII转化活性。我们最近 发现mtrII能够转化人表皮细胞， 角质形成细胞系（RHEK-1）的致瘤性，并将肿瘤 诊断为低分化癌。 进一步的实验将 有必要确定ORF在转化中的作用， 产生进一步的缺失克隆或突变ORF。也 将尝试绘制转录本，以确定 同一基因在转化细胞和感染细胞中表达。 关于mtrIII（7.5kb）转化结构域，CMV主要直接转化结构域（mtrIII）是CMV主要直接转化结构域。 早期基因（IE-1）。该IE-1基因是一个候选基因。 亚单位疫苗我们的缺失分析定位了 在IE-1基因以外的2.1kb区域具有mtrIII活性。这个区域也 包含了几个ORF，目前正在调查他们的 转型中的角色。 这些研究将适用于产生安全的减毒活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.