MECHANISTIC APPROACHES TO HCMV MTRII-INDUCED TRANSFORMATION

已知人类疱疹病毒与致癌性 疾病我们以前报道过HCMV株Towne的两个区域可以 肿瘤转化NIH 3 T3和Rat-2细胞。这些是XbaI/BamHI EM （mtrII）和Xba I/BamHI EJ（mtrIII）。mtrII区域包含三个开放的 79、83和34个氨基酸的阅读框（ORF）。在目前的研究中，我们 主要集中在mtrII序列的功能分析， 我想知道mtrII ORF是否负责转化。我们产生 通过DNA检测具有新霉素抗性标记的稳定mtrII转化细胞系 转染NIH 3 T3细胞。这些细胞系在小鼠中产生肿瘤。 通过北方印迹分析，我们一致地检测到mtrII特异性RNA， 这些转化体。然后我们询问mtrII是否包含任何启动子序列 这些成绩单氯霉素乙酰转移酶（CAT） 在Cos-7细胞中，我们检测到上游的弱启动子活性， 当以有义方向与CAT基因连接时，mtrII的285 bp区域 关于ORFs 然而，整个980 bp的mtrII区域没有 可检测的启动子活性，而不管方向如何。285 bp 区域也有弱的转录增强子活性， 导向用CV-1细胞获得了类似的结果。这些研究 在mtrII中建立DNA序列可以顺式激活基因表达， 位于调节mtrII ORFs表达的位置。 然后，我们检查了CMV立即早期（IE）基因和HIV TAT基因是否与CMV感染有关。 基因可以调控mtrII的启动子活性。没有反式激活 启动子序列用HIV达特基因或CMV IE基因检测。这些 研究表明，在很大程度上， 控制mtrII启动子活性。进一步的研究将解决起源 这些成绩单，并评估的作用，个别ORF在mtrII 介导转化这些研究将有助于发展 一个安全的候选疫苗对抗人类巨细胞病毒。该项目仍在进行中。

Human herpeviruses have been known to be associated with oncogenic diseases. We previously reported that two regions of HCMV strain Towne can neoplastically transform NIH 3T3 and Rat-2 cells. These are XbaI/BamHI EM (mtrII) and XbaI/BamHI EJ (mtrIII). The mtrII region contains three open reading frames (ORF) of 79, 83 and 34 amino acids. In the current study, we primarily focused on the functional analysis of the mtrII sequence and wondered if mtrII ORFs are responsible for transformation. We generated stable mtrII transformed cell lines with neomycin resistant marker by DNA transfection into NIH 3T3 cells. These cell lines produced tumors in mice. By Northern blot analysis, we consistently detected mtrII specific RNAs in these transformants. We then asked if mtrII contains any promoter sequence for these transcripts. Using chloramphenicol acetyl transferase (CAT) assays in Cos-7 cells, we detected weak promoter activity in the upstream 285 bp region of mtrII when linked to CAT gene in the sense orientation with respect to ORFS. However, the entire 980 bp mtrII region had no detectable promoter activity regardless of the orientation. The 285 bp region also had weak transcriptional enhancer activity only in the sense orientation. Similar results were obtained with CV-1 cells. These studies establish that DNA sequences in mtrII can cis-activate gene expression and are in a location to regulate the expression of the mtrII ORFS. We then examined whether the CMV immediate-early (IE) genes and the HIV TAT gene can regulate the promoter activity in mtrII. No transactivation of the promoter sequence was detected by HIV TAT gene or CMV IE genes. These studies indicate that cellular transcription factors, in large part, control mtrII promoter activity. Further studies would resolve the origin of these transcripts and assess the role of the individual ORFs in mtrII mediated transformation. These studies would be useful to the development of a safe candidate vaccine against human CMV. The project is still active.