Computational and molecular analysis of conserved influenza A virus RNA secondary structures involved in infectious virion production
Computational and molecular analysis of conserved influenza A virus RNA secondary structures involved in infectious virion production
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DOI：
10.1080/15476286.2016.1208331
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发表时间：
2016-01-01
期刊：
影响因子：
4.1
通讯作者：
Pybus, Oliver G.
中科院分区：
文献类型：
--
作者：
Kobayashi, Yuki;Dadonaite, Bernadeta;Pybus, Oliver G.
除了编码病毒蛋白，RNA病毒的基因组还具有二级和三级RNA结构，这些结构与成功复制和繁殖所必需的功能相关。在这里，我们使用计算和进化方法鉴定了来自不同亚型和宿主物种的甲型流感病毒（IAV）毒株的1，884 M片段序列中极其保守的茎环结构。这些结构分别在M1和M2的编码区的3端和5端预测，其中先前已经提出存在包装信号。这些信号被认为是将8个不同负链RNA片段（vRNA）的单拷贝掺入IAV颗粒所必需的。为了直接测试保守的茎环结构的功能，我们进行了反向遗传实验，以引入设计为破坏在3个位置预测的二级结构的同义突变，并发现它们减弱重组病毒的感染性。在一个突变体中，预测在核苷酸位置219-240处破坏茎环结构，在升高的温度下减毒更明显，并且伴随有缺陷的病毒颗粒的产生增加。我们的研究结果表明，保守的二级结构中预测的M段参与生产的传染性病毒颗粒在IAV复制。
As well as encoding viral proteins, genomes of RNA viruses harbor secondary and tertiary RNA structures that have been associated with functions essential for successful replication and propagation. Here, we identified stem-loop structures that are extremely conserved among 1,884M segment sequences of influenza A virus (IAV) strains from various subtypes and host species using computational and evolutionary methods. These structures were predicted within the 3 and 5 ends of the coding regions of M1 and M2, respectively, where packaging signals have been previously proposed to exist. These signals are thought to be required for the incorporation of a single copy of 8 different negative-strand RNA segments (vRNAs) into an IAV particle. To directly test the functionality of conserved stem-loop structures, we undertook reverse genetic experiments to introduce synonymous mutations designed to disrupt secondary structures predicted at 3 locations and found them to attenuate infectivity of recombinant virus. In one mutant, predicted to disrupt stem loop structure at nucleotide positions 219-240, attenuation was more evident at increased temperature and was accompanied by an increase in the production of defective virus particles. Our results suggest that the conserved secondary structures predicted in the M segment are involved in the production of infectious viral particles during IAV replication.