Generation of infectious RNA complexes in Orbiviruses: RNA-RNA interactions of genomic segments.
Generation of infectious RNA complexes in Orbiviruses: RNA-RNA interactions of genomic segments.
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DOI：
10.18632/oncotarget.12496
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发表时间：
2016-11-08
期刊：
影响因子：
--
通讯作者：
Roy P
中科院分区：
文献类型：
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作者：
Fajardo T Jr;AlShaikhahmed K;Roy P
具有节段化RNA基因组的病毒必须包装正确数量的片段以合成具有感染性的病毒颗粒。最近的研究表明，具有节段化双链RNA基因组的呼肠孤病毒科家族成员通过在被招募到组装衣壳之前形成片段RNA网络来完成这一具有挑战性的任务，尽管仍然缺乏直接证据。在这里，我们研究了呼肠孤病毒科成员H病毒(EHDV)10个RNA片段的预制复合体的病毒恢复能力，方法是在单个体外反应中转录基因组RNA片段的准确T7cDNA拷贝，然后转染哺乳动物细胞。得到的数据进一步证实了蓝舌病毒产生的RNA复合体，蓝舌病病毒是另一个家族成员。用蔗糖梯度超速离心法证明了RNA复合体的形成，用凝胶迁移率改变分析证实了RNA复合体的形成所固有的RNA-RNA相互作用。此外，我们还表明，破坏RNA复合体的形成会抑制病毒的恢复，这证实了完整的RNA网络的招募对于包装和因此的病毒恢复是必不可少的。这种高效的反向遗传学系统将有助于进一步了解呼肠孤病毒科成员的进化关系，并可能有助于抗病毒分子的开发。
Viruses with segmented RNA genomes must package the correct number of segments for synthesis of infectious virus particles. Recent studies suggest that the members of the Reoviridae family with segmented double-stranded RNA genomes achieve this challenging task by forming RNA networks of segments prior to their recruitment into the assembling capsid albeit direct evidence is still lacking. Here, we investigated the capability of virus recovery by preformed complexes of ten RNA segments of H Virus (EHDV), a Reoviridae member, by transcribing exact T7 cDNA copies of genomic RNA segments in a single in vitro reaction followed by transfection of mammalian cells. The data obtained was further confirmed by RNA complexes generated from Bluetongue virus, another family member. Formation of RNA complexes was demonstrated by sucrose gradient ultracentrifugation, and RNA-RNA interactions inherent to the formation of the RNA complexes were demonstrated by electrophoretic mobility shift assay. Further, we showed that disruption of RNA complex formation inhibits virus recovery, confirming that recruitment of complete RNA networks is essential for packaging and consequently, virus recovery. This efficient reverse genetics system will allow further understanding of evolutionary relationships of Reoviridae members and may also contribute to development of antiviral molecules.