Norton P A, Coffin J M
J Virol. 1987 Apr;61(4):1171-9. doi: 10.1128/JVI.61.4.1171-1179.1987.
Using the Escherichia coli lacZ gene product beta-galactosidase as an indicator of gene expression, we analyzed sequences that are required for expression of the Rous sarcoma virus (RSV) genome in avian cells. The RSV long terminal repeat (LTR) and leader region were sufficient to direct the synthesis of high levels of enzymatically active gag-lacZ fusion proteins. A portion of U3 greater than 140 nucleotides upstream from the cap site was essential for gene expression. This element functioned in either orientation, but its activity was attenuated when it was relocated further away from the cap site. The insertion of exogenous LTRs 3' of lacZ augmented the expression of that gene by increasing the level of stable gag-lacZ transcripts. Furthermore, 3' LTRs could partially compensate for certain defects within the 5' LTR. Insertion of various fragmentary LTRs allowed the identification of at least three synergistically acting domains within the 3' LTR that influence gene expression. Interestingly, the gag-lacZ expression was only stimulated by a 3' LTR when the exogenous 3'-untranslated region was adjacent. Our results imply that the two LTRs of a provirus interact in a complex manner to promote high levels of stable transcripts. It was also found that gag-lacZ expression was independent of viral gene products, suggesting that trans-activation is not a key mechanism regulating RSV expression in avian cells.
我们以大肠杆菌乳糖操纵子的β-半乳糖苷酶基因产物作为基因表达的指标，分析了禽细胞中劳斯肉瘤病毒（RSV）基因组表达所需的序列。RSV长末端重复序列（LTR）和前导区足以指导高水平的具有酶活性的gag-lacZ融合蛋白的合成。帽位点上游大于140个核苷酸的U3部分对于基因表达至关重要。该元件在任一方向均可发挥作用，但其活性在远离帽位点时会减弱。lacZ 3'端插入外源LTR通过增加稳定的gag-lacZ转录本水平增强了该基因的表达。此外，3' LTR可以部分弥补5' LTR内的某些缺陷。插入各种片段化的LTR使得能够鉴定出3' LTR内至少三个协同作用的影响基因表达的结构域。有趣的是，只有当外源3'-非翻译区相邻时，gag-lacZ表达才会受到3' LTR的刺激。我们的结果表明，前病毒的两个LTR以复杂的方式相互作用以促进高水平的稳定转录本。还发现gag-lacZ表达独立于病毒基因产物，这表明反式激活不是调节禽细胞中RSV表达的关键机制。
