Metal ion catalysis of RNA cleavage by the influenza virus endonuclease
Metal ion catalysis of RNA cleavage by the influenza virus endonuclease
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DOI：
10.1021/bi9828932
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发表时间：
1999-04-27
期刊：
影响因子：
2.9
通讯作者：
Klumpp, K
中科院分区：
文献类型：
--
作者：
Doan, L;Handa, B;Klumpp, K
流感病毒依赖RNA的RNA聚合酶蛋白复合体包含相关的RNA内切酶活性，它在帽结构下游9-15个核苷酸的特定位置切割细胞核中的宿主mRNA前体。这一反应提供了封端的寡核苷酸，其功能是启动病毒mRNA的合成。内切酶反应依赖于二价金属离子的存在。我们使用了一些二价和三价金属离子单独和病态结合来探索流感病毒核酸内切酶切割RNA的机制。不同金属离子对病毒有较高的切割活性，其中以100 mU M Mn2+或100 mU M Co2+的切割活性最高。在最佳浓度为1 mM时，该酶活约为添加镁离子时的2倍。活性依赖于金属离子浓度，Hill系数接近或大于2。不同浓度的不同金属离子组合对切割活性有协同激活作用。这些结果支持流感病毒帽依赖内切酶的RNA裂解的双金属离子机制。这些发现也与聚合酶的结构模型一致，在该模型中，特定的核酸内切酶活性部位与聚合酶模块的核苷酸转移酶活性部位在空间上是分开的。
The influenza virus RNA-dependent RNA polymerase protein complex contains an associated RNA endonuclease activity, which cleaves host mRNA precursors in the cell nucleus at defined positions 9-15 nucleotides downstream of the cap structure. This reaction provides capped oligoribonucleotides, which function as primers for the initiation of viral mRNA synthesis. The endonuclease reaction is dependent on the presence of divalent metal ions. We have used a number of divalent and trivalent metal ions alone and ill combination to probe the mechanism of RNA cleavage by the influenza virus endonuclease. Virus-specific cleavage was observed with various metal ions, and maximum cleavage activity was obtained with 100 mu M Mn2+ or 100 mu M Co2+. This activity was about 2-fold higher than that observed with Mg2+ at the optimal concentration of 1 mM. Activity dependence on metal ion concentration was cooperative with Hill coefficients close to or larger than 2. Synergistic activation of cleavage activity was observed with combinations of different metal ions at varying concentrations. These results support a two-metal ion mechanism of RNA cleavage for the influenza virus cap-dependent endonuclease. The findings are also consistent with a structural model of the polymerase, in which the specific endonuclease active site is spatially separated from the nucleotidyl transferase active site of the polymerase module.