Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo
Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo
复制标题
DOI：
10.1016/j.virol.2003.08.039
复制
发表时间：
2003-12-20
期刊：
影响因子：
3.7
通讯作者：
Nagy, PD
中科院分区：
文献类型：
--
作者：
Panaviene, Z;Nagy, PD
RNA重组是病毒进化的主要驱动力之一，被认为是由于病毒复制过程中的复制酶错误而发生的。在本文中，我们证明了黄瓜坏死病毒(一种番茄病毒)的复制酶蛋白直接参与体内的RNA重组。复制酶蛋白质的RNA结合域中的突变影响了用典型的缺陷干扰(DI)RNA观察到的重组频率，DI RNA是重组研究的模型模板。与野生型辅助病毒相比，测试的17个复制酶突变体中有5个重组体的形成延迟。有趣的是，两个复制酶突变体加速了重组的形成，此外，这些突变体还增加了亚基因组RNA合成的水平(病毒学308(2003)，191-205)。陷阱互补系统被用来证明p33复制酶蛋白的突变导致重组率改变。分离的重组子大多是不精确的(非同源的)，重组位点分别聚集在复制增强子区和假定的顺式作用元件周围。这些RNA元件可能通过番茄病毒复制酶促进所提出的模板切换事件。结合上面引用的文章中的数据，这里提出的结果坚定地证明了复制酶蛋白质的保守的RNA结合基序参与了RNA复制、亚基因组RNA合成和RNA重组。(C)2003 Elsevier Inc.保留所有权利。
RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A traps-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination. (C) 2003 Elsevier Inc. All rights reserved.