Rice S A, Klessig D F
J Virol. 1985 Dec;56(3):767-78. doi: 10.1128/JVI.56.3.767-778.1985.
A genetic system is described which allows the isolation and propagation of adenovirus mutants containing lesions in early region 2A (E2A), the gene encoding the multifunctional adenovirus DNA-binding protein (DBP). A cloned E2A gene was first mutagenized in vitro and then was introduced into the viral genome by in vivo recombination. The E2A mutants were propagated by growth in human cell lines which express an integrated copy of the DBP gene under the control of a dexamethasone-inducible promoter (D. F. Klessig, D. E. Brough, and V. Cleghon, Mol. Cell. Biol. 4:1354-1362, 1984). The protocol was used to construct five adenovirus mutants, Ad5d1801 through Ad5d1805, which contained deletions in E2A. One of the mutants, Ad5d1802, made no detectable DBP and thus represents the first DBP-negative adenovirus mutant, while the four other mutants made truncated DBP-related polypeptides. All five mutants were completely defective for growth and plaque formation on HeLa cell monolayers. Furthermore, the two mutants which were tested, Ad5d1801 and Ad5d1802, did not replicate their DNA in HeLa cells. The mutant Ad5d1804 encoded a truncated DBP-related protein which contained an entire amino-terminal domain derived from the host range mutant Ad5hr404, a variant of Ad5 which multiplies efficiently in monkey cells. While results of a previous study suggest that the amino-terminal domain of DBP could act independently of the carboxyl-terminal domain to enhance late gene expression in monkey cells, the Ad5d1804 polypeptide failed to relieve the block to late viral protein synthesis in monkey cells. The mutant Ad5d1802 was used to study the role of DBP in the regulation of early adenovirus gene expression in infected HeLa cells. These experiments show that E2A mRNA levels are consistently reduced approximately fivefold in Ad5d1802-infected cells, suggesting either a role for DBP in the expression of its own gene or a cis-acting defect caused by the E2A deletion. DBP does not appear to play a significant role in the regulation of adenovirus early regions 1A, 1B, 3, or 4 mRNA levels in infected HeLa cell monolayers since wild-type Ad5- and Ad5d1802-infected cells showed very little difference in the patterns of expression of these genes.
本文描述了一种遗传系统，该系统可用于分离和繁殖在早期区域2A（E2A）中存在损伤的腺病毒突变体，E2A基因编码多功能腺病毒DNA结合蛋白（DBP）。首先在体外对克隆的E2A基因进行诱变，然后通过体内重组将其引入病毒基因组。E2A突变体通过在人细胞系中生长来繁殖，这些细胞系在地塞米松诱导型启动子的控制下表达DBP基因的整合拷贝（D.F.克莱西格、D.E.布劳和V.克莱贡，《分子细胞生物学》4:1354 - 1362，1984）。该方案用于构建五个腺病毒突变体，即Ad5d1801至Ad5d1805，它们在E2A中存在缺失。其中一个突变体Ad5d1802未检测到DBP，因此代表首个DBP阴性腺病毒突变体，而其他四个突变体产生截短的DBP相关多肽。所有五个突变体在HeLa细胞单层上的生长和噬斑形成均完全缺陷。此外，所测试的两个突变体Ad5d1801和Ad5d1802在HeLa细胞中不复制其DNA。突变体Ad5d1804编码一种截短的DBP相关蛋白，该蛋白包含来自宿主范围突变体Ad5hr404的完整氨基末端结构域，Ad5hr404是Ad5的一个变体，能在猴细胞中高效繁殖。虽然先前一项研究的结果表明DBP的氨基末端结构域可独立于羧基末端结构域发挥作用，以增强猴细胞中的晚期基因表达，但Ad5d1804多肽未能解除对猴细胞中晚期病毒蛋白合成的阻断。突变体Ad5d1802用于研究DBP在感染的HeLa细胞中腺病毒早期基因表达调控中的作用。这些实验表明，在Ad5d1802感染的细胞中，E2A mRNA水平持续降低约五倍，这表明DBP可能在其自身基因的表达中起作用，或者是由E2A缺失导致的顺式作用缺陷。由于野生型Ad5和Ad5d1802感染的细胞在这些基因表达模式上差异很小，因此DBP似乎在感染的HeLa细胞单层中腺病毒早期区域1A、1B、3或4 mRNA水平的调控中不发挥重要作用。
