Song B, Young C S
Department of Microbiology, Columbia University, New York, New York 10032, USA.
J Virol. 1998 Apr;72(4):3213-20. doi: 10.1128/JVI.72.4.3213-3220.1998.
Comparisons among sequences predicted to encode the major late promoter (MLP) of adenoviruses from a wide variety of host species show that an inverted CAAT box is among the most highly conserved transcription elements found in the putative MLPs. The high degree of conservation suggests that the CAAT box plays an important role in the function of the MLP in vivo, an idea supported by a previous mutational analysis of the core CCAAT sequence. To address the importance of the CAAT box, in terms both of quantitative levels of transcription and of specificity, a further set of mutations was created and examined in the context of the viral genome. One mutation, CAAT5, contains individual changes at five positions, four of which correspond to invariant residues in a CAAT box consensus derived either by computer analysis or empirically. The CAAT5 mutation had no discernible phenotype by itself but when coupled with the previously described USF0 mutation, which disrupts binding of the upstream stimulating factor (USF) but is otherwise phenotypically silent, gave rise to virus with a severe replication deficiency. Nuclear run-on assays showed that transcription initiation at the mutant MLP was significantly reduced compared with that of the wild type or the virus containing CAAT5 alone. Replication of the double mutant was lower than that of the previously described USF0::CCCAT virus, suggesting that the additional mutations in the CAAT box had further lowered the binding of transcription factor CP1 (also called CBF, NF-Y). Replacement of the CAAT box by an ATF binding site or an OCT1 binding site had no phenotypic effect in an otherwise wild-type background, but replacement in a USF0::CCCAT background led to only partial restoration of the wild-type phenotype. The failure to restore the functional redundancy normally exhibited by the CAAT box and the proximal upstream activating element is consistent with the idea that in the adenovirus MLP the CAAT box is preferred over others as the distal transcriptional element.
对来自多种宿主物种的腺病毒主要晚期启动子（MLP）预测编码序列的比较表明，反向CAAT框是在假定的MLP中发现的最高度保守的转录元件之一。高度的保守性表明CAAT框在体内MLP的功能中起重要作用，这一观点得到了先前对核心CCAAT序列的突变分析的支持。为了从转录定量水平和特异性两方面探讨CAAT框的重要性，在病毒基因组背景下创建并检测了另一组突变。一种突变体CAAT5在五个位置有单独的变化，其中四个对应于通过计算机分析或经验推导的CAAT框共有序列中的不变残基。CAAT5突变本身没有可察觉的表型，但当与先前描述的USF0突变结合时，USF0突变破坏上游刺激因子（USF）的结合但在其他方面表型沉默，产生了具有严重复制缺陷的病毒。核转录分析表明，与野生型或仅含CAAT5的病毒相比，突变MLP处的转录起始显著减少。双突变体的复制低于先前描述的USF0::CCCAT病毒，这表明CAAT框中的额外突变进一步降低了转录因子CP1（也称为CBF、NF-Y）的结合。在其他方面为野生型背景下，用ATF结合位点或OCT1结合位点取代CAAT框没有表型效应，但在USF0::CCCAT背景下取代仅导致野生型表型的部分恢复。未能恢复CAAT框和近端上游激活元件通常表现出的功能冗余，这与腺病毒MLP中CAAT框作为远端转录元件比其他元件更受青睐的观点一致。
