A compensatory mutagenesis study of a conserved hairpin in the M gene segment of influenza A virus shows its role in virus replication.
A compensatory mutagenesis study of a conserved hairpin in the M gene segment of influenza A virus shows its role in virus replication.
复制标题
对流感A基因段中保守的发夹的补偿性诱变研究显示了其在病毒复制中的作用。
DOI：
10.1080/15476286.2017.1338243
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发表时间：
2017-11-02
期刊：
影响因子：
4.1
通讯作者：
Gultyaev AP
中科院分区：
文献类型：
--
作者：
Spronken MI;van de Sandt CE;de Jongh EP;Vuong O;van der Vliet S;Bestebroer TM;Olsthoorn RCL;Rimmelzwaan GF;Fouchier RAM;Gultyaev AP
人们越来越认识到RNA结构在甲型流感病毒复制过程中的重要性。在这里，我们研究了在vRNA5‘包装信号区的M基因片段(NT 967-994)中预测的保守发夹。用补偿突变方法研究了这种RNA结构的存在及其在病毒复制中的可能作用。在发夹茎中引入了基于自然变异的突变。对RNA结构破坏和恢复的突变病毒的病毒复制特性进行了研究。与野生型(WT)病毒相比，具有结构破坏突变的病毒具有更低的病毒滴度和显著的中值空斑大小，而具有结构恢复突变的病毒复制能力与野生型相当。此外，当发夹环中引入突变时，病毒复制也减少，这表明它参与了RNA相互作用。Northern印迹和FACS实验研究了通过选择性剪接表达的RNA水平以及M1和M2蛋白生产的差异。茎破坏突变导致vRNA和M2 mRNA水平降低，并在早期时间点减少M2蛋白的产生。当RNA结构恢复时，vRNA、M2 mRNA和M2蛋白水平升高，表现出代偿效应。因此，本研究为预测的M RNA结构的功能重要性提供了证据，并表明其在剪接调控中的作用。
RNA structures are increasingly recognized to be of importance during influenza A virus replication. Here, we investigated a predicted conserved hairpin in the M gene segment (nt 967-994) within the region of the vRNA 5′ packaging signal. The existence of this RNA structure and its possible role in virus replication was investigated using a compensatory mutagenesis approach. Mutations were introduced in the hairpin stem, based on natural variation. Virus replication properties were studied for the mutant viruses with disrupted and restored RNA structures. Viruses with structure-disrupting mutations had lower virus titers and a significantly reduced median plaque size when compared with the wild-type (WT) virus, while viruses with structure restoring-mutations replicated comparable to WT. Moreover, virus replication was also reduced when mutations were introduced in the hairpin loop, suggesting its involvement in RNA interactions. Northern blot and FACS experiments were performed to study differences in RNA levels as well as production of M1 and M2 proteins, expressed via alternative splicing. Stem-disruptive mutants caused lower vRNA and M2 mRNA levels and reduced M2 protein production at early time-points. When the RNA structure was restored, vRNA, M2 mRNA and M2 protein levels were increased, demonstrating a compensatory effect. Thus, this study provides evidence for functional importance of the predicted M RNA structure and suggests its role in splicing regulation.
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影响因子：
5.8
作者：
Gultyaev, Alexander P.;Heus, Hans A.;Olsthoorn, Rene C. L.
通讯作者：
Olsthoorn, Rene C. L.
DOI：
10.1073/pnas.84.22.8140
发表时间：
1987-11-01
影响因子：
11.1
作者：
HSU, MT;PARVIN, JD;PALESE, P
通讯作者：
PALESE, P
DOI：
10.3390/v8060165
发表时间：
2016-06-17
期刊：
Viruses
影响因子：
--
作者：
Giese S;Bolte H;Schwemmle M
通讯作者：
Schwemmle M
DOI：
10.1038/nrmicro3367
发表时间：
2015-01
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
Eisfeld AJ;Neumann G;Kawaoka Y
通讯作者：
Kawaoka Y
DOI：
10.1073/pnas.1308649110
发表时间：
2013-10-01
影响因子：
11.1
作者：
Essere, Boris;Yver, Matthieu;Moules, Vincent
通讯作者：
Moules, Vincent