MECHANISTIC APPROACHES TO HCMV MTRII AND MTRIII-INDUCED TRANSFORMATION

HCMV MTRII 和 MTRIII 诱导转化的机制方法

• 批准号: 3792524
• 负责人: A RAZZAQUE
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3792524
• 关键词: 3T3 cells; Herpesviridae vaccine; cell transformation; chloramphenicol acetyltransferase; clone cells; cytomegalovirus; gene deletion mutation; gene expression; genetic mapping; genetic promoter element; genome; human tissue; keratinocyte; laboratory mouse; messenger RNA; molecular cloning; nucleic acid sequence; oncogenic virus; open reading frames; regulatory gene; site directed mutagenesis; transfection; transforming virus; viral carcinogenesis; virus genetics

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.

人类巨细胞病毒与多种类型的人类 恶性疾病，如神经母细胞瘤、前列腺癌、宫颈癌 癌症和结肠癌。病毒在艾滋病和艾滋病中被重新激活 移植病人。它的转化潜力令人非常关注 在疫苗研发方面。我们在Long中报道了两个转换结构域 人巨细胞病毒基因组的独特片段：I)mtrII(980bp) 和ii)MTRIII(7.5kb)。对变换函数进行了研究 产生缺失克隆，通过DNA介导的基因转染克隆 转移至小鼠3T3或大鼠-2细胞系，并对其进行检测 琼脂糖中非锚定生长和对小鼠的致瘤性。 MtrII区被发现包含3个开放阅读框(ORF)(79， 83和34个氨基酸)进行DNA序列分析。我们还报道了发起人 用CAT分析和RT-PCR方法研究MTRII中ORF上游区域的活性 转化子中mtrII转录本的检测。相似的mRNA 在HCMV感染的细胞中也检测到了种类。沿着这些思路， 在人类细胞中也检测了mtrII的转化活性。我们最近 鉴定mtrII能够转化人的表皮 角质形成细胞系(RHEK-1)对其致瘤性的影响 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在转换中的作用 产生进一步的缺失克隆或ORF的突变。也是 将尝试对成绩单进行映射，以确定是否 在转化细胞和感染细胞中都表达相同的基因。 关于MTRIII(7.5kb)的转换结构域，CMV主要立即 其中含有早期基因(IE-1)。这个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转换 MtrIII的活性位于IE-1基因之外的2.1kb区域。这一地区也 包含几个ORF，这些ORF目前正在接受调查 在转型中扮演的角色。 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。