Stunnenberg H G, Lange H, Philipson L, van Miltenburg R T, van der Vliet P C
European Molecular Biology Laboratory, Heidelberg FRG.
Nucleic Acids Res. 1988 Mar 25;16(6):2431-44. doi: 10.1093/nar/16.6.2431.
Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system.
腺病毒（Ad）DNA复制的起始通过蛋白质引发机制发生，该机制需要病毒前体末端蛋白（pTP）和DNA聚合酶（pol）以及来自未感染HeLa细胞的两种核DNA结合蛋白。由于pTP和DNA聚合酶蛋白丰度低且在Ad感染细胞中以pTP-pol复合物形式存在，分别对它们进行生化研究受到了阻碍。我们构建了一个基因组序列，其中包含来自Ad5 pol基因的大开放阅读框，并连接了来自一个推定外显子的9个碱基对。当插入痘苗病毒的修饰晚期启动子后面时，产生的重组病毒产生了具有酶活性的140 kDa Ad DNA聚合酶。同样的策略被用于以功能形式表达80 kDa pTP基因。与腺病毒感染细胞的提取物相比，这两种蛋白均过表达至少30倍，并且在体外腺病毒DNA复制系统中结合时，它们对起始反应具有完全活性。
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