Mutations in active-site residues of the uracil-DNA glycosylase encoded by vaccinia virus are incompatible with virus viability
Mutations in active-site residues of the uracil-DNA glycosylase encoded by vaccinia virus are incompatible with virus viability
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DOI：
10.1128/jvi.70.11.7965-7973.1996
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发表时间：
1996-11-01
影响因子：
5.4
通讯作者：
McFadden, G
中科院分区：
文献类型：
--
作者：
Ellison, KS;Peng, W;McFadden, G
痘苗病毒的 D4R 基因编码对病毒活力至关重要的功能性尿嘧啶-DNA 糖基化酶（D. T. Stuart、C. Upton、M. A. Eligman、E. G. Niles 和 G. McFadden, J. Virol. 67:2503-2513, 1993），并且 D4R 突变体 ts4149 赋予条件性致死缺陷(A.K.Millns、M.S.Carpenter 和 A.M.DeLange, Virology 198:504-513, 1994)。突变体 ts4149 蛋白在体外表达并测定尿嘧啶-DNA 糖基化酶活性。在允许的温度下观察到野生型活性不到 6%，但 ts4149 蛋白在非允许的温度下完全失活。将 ts4149 基因诱变回野生型（Arg-179-->Gly）恢复了全部活性。在允许的温度下感染细胞的裂解物中，ts4149蛋白显着减少，并且即使在尿嘧啶糖基化酶抑制剂蛋白存在的情况下也检测不到其活性，尿嘧啶糖基化酶抑制剂蛋白抑制宿主尿嘧啶-DNA糖基化酶，但不抑制痘苗病毒。因此，ts4149 蛋白不耐热，将尿嘧啶去除与痘苗病毒 DNA 复制相关。痘苗病毒尿嘧啶-DNA糖基化酶的三个活性位点氨基酸发生突变（Asp-68-->Asn、Asn-120-->Val和His-181-->Leu），产生尿嘧啶切除完全缺陷但仍保留结合DNA能力的蛋白质。将每个突变的 D4R 基因转染到痘苗病毒 ts4149 感染的细胞中，以评估允许病毒在 40 摄氏度下存活的重组事件。遗传分析和测序研究表明，唯一存活的病毒是重组消除了突变位点的病毒。我们得出的结论是，D4R 蛋白的尿嘧啶裂解活性对其在痘苗病毒 DNA 复制中的功能至关重要，这表明尿嘧啶残基的去除起着必然的作用。
The D4R gene of vaccinia virus encodes a functional uracil-DNA glycosylase that is essential for viral viability (D. T. Stuart, C. Upton, M. A. Eligman, E. G. Niles, and G. McFadden, J. Virol. 67:2503-2513, 1993), and a D4R mutant, ts4149, confers a conditional lethal defect in viral DNA replication (A. K. Millns, M. S. Carpenter, and A. M. DeLange, Virology 198:504-513, 1994). The mutant ts4149 protein was expressed in vitro and assayed for uracil-DNA glycosylase activity. Less than 6% of wild-type activity was observed at permissive temperatures, but the ts4149 protein was completely inactive at the nonpermissive temperature. Mutagenesis of the ts4149 gene back to wild type (Arg-179-->Gly) restored full activity. The ts4149 protein was considerably reduced in lysates of cells infected at the permissive temperature, and its activity was undetectable, even in the presence of the uracil glycosylase inhibitor protein, which inhibits the host uracil-DNA glycosylases but not that of vaccinia virus. Thus the ts4149 protein is thermolabile, correlating uracil removal with vaccinia virus DNA replication. Three active-site amino acids of the vaccinia virus uracil-DNA glycosylase were mutated (Asp-68-->Asn, Asn-120-->Val, and His-181-->Leu), producing proteins that were completely defective in uracil excision but still retained the ability to bind DNA. Each mutated D4R gene was transfected into vaccinia virus ts4149-infected cells in order to assess the recombination events that allowed virus survival at 40 degrees C. Genetic analysis and sequencing studies revealed that the only viruses to survive were those in which recombination eliminated the mutant locus. We conclude that the uracil cleavage activity of the D4R protein is essential for its function in vaccinia virus DNA replication, suggesting that the removal of uracil residues plays an obligatory role.