Rajan P, Dhamankar V, Rundell K, Thimmapaya B
Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, Illinois 60611-3008.
J Virol. 1991 Dec;65(12):6553-61. doi: 10.1128/JVI.65.12.6553-6561.1991.
Transcriptional stimulatory properties of virus-encoded transactivators appear to be critical for viral gene expression and may be linked to cellular transformation in certain cases. Recently, the simian virus 40 (SV40) 17-kDa small-t antigen was shown to stimulate transcription of polymerase II and III genes in transient transfection assays. In experiments performed in our laboratory, two of the polymerase II promoters of the adenovirus genome, namely, the EII-early and EIII promoters, were transactivation, we examined the transient transfection assays. To further elucidate the mechanism of this transactivation, we examined the ability of small-t to transactivate the adenovirus type 5 EII-early and EIII promoters in CV-1 cells under conditions in which the small-t gene or the reporter genes were introduced into the cells through transfection and other routes. In one approach, we used established CV-1 cell lines which constitutively express the small-t gene, and study of the EII-early promoter was afforded by infection of an EIA-negative adenovirus type 5 variant. For the second approach, a recombinant adenovirus was constructed in which small-t was expressed from a replication origin-negative SV40 early promoter in the EIA region of an adenovirus vector (Ad-SV-t). The effect of small-t on adenovirus EII-early and EIII promoter expression was studied in coinfection or single-infection experiments. In both cases, transcription of the adenovirus early promoters was not stimulated by small-t. These and other results indicate that transactivation of polymerase II promoters by small-t occurs only when the target gene is in a transiently transfected state. Thus, small-t-mediated transactivation of polymerase II promoters is dependent on the type of assay system used and may be mechanistically different from that of the widely studied EIA.
病毒编码的反式激活因子的转录刺激特性对于病毒基因表达似乎至关重要，并且在某些情况下可能与细胞转化有关。最近，在瞬时转染实验中，猿猴病毒40（SV40）17-kDa小t抗原被证明可刺激聚合酶II和III基因的转录。在我们实验室进行的实验中，腺病毒基因组的两个聚合酶II启动子，即EII-早期启动子和EIII启动子，被反式激活，我们检测了瞬时转染实验。为了进一步阐明这种反式激活的机制，我们检测了在通过转染和其他途径将小t基因或报告基因导入细胞的条件下，小t在CV-1细胞中反式激活5型腺病毒EII-早期和EIII启动子的能力。在一种方法中，我们使用了组成性表达小t基因的已建立的CV-1细胞系，并通过感染EIA阴性的5型腺病毒变体来研究EII-早期启动子。对于第二种方法，构建了一种重组腺病毒，其中小t由腺病毒载体（Ad-SV-t）的EIA区域中来自复制起点阴性的SV40早期启动子表达。在共感染或单感染实验中研究了小t对腺病毒EII-早期和EIII启动子表达的影响。在这两种情况下，腺病毒早期启动子转录均未被小t刺激。这些以及其他结果表明，只有当靶基因处于瞬时转染状态时，小t才会反式激活聚合酶II启动子。因此，小t介导的聚合酶II启动子反式激活取决于所使用的检测系统类型，并且在机制上可能与广泛研究的EIA不同。
