Boyce Mark, McCrae Malcom A, Boyce Paul, Kim Jan T
The Pirbright Institute, Pirbright, Woking GU24 0NF, UK.
Mott MacDonald, Mott MacDonald House, 8-10 Sydenham Road, Croydon, CR0 2EE.
J Gen Virol. 2016 May;97(5):1145-1157. doi: 10.1099/jgv.0.000400. Epub 2016 Jan 12.
The process by which eukaryotic viruses with segmented genomes select a complete set of genome segments for packaging into progeny virus particles is not understood. In this study a model based on the association of genome segments through specific RNA-RNA interactions driven by base pairing was formalized and tested in the Orbivirus genus of the Reoviridae family. A strategy combining screening of the genomic sequences for inter-segment complementarity with direct functional testing of inter-segment RNA-RNA interactions using reverse genetics is described in the type species of the Orbivirus genus, Bluetongue virus (BTV). Two examples, involving four of the ten BTV genomic segments, of specific inter-segment interaction motifs whose maintenance is essential for the generation of infectious virus, were identified. Equivalent inter-segment complementarities were found between the identified regions of the orthologous genome segments of all orbiviruses, including phylogenetically distant species. Specific interaction of the participating RNA segments was confirmed in vitro using electrophoretic mobility shift assays, with the interactions inhibited using oligonucleotides complementary to the interaction motif of one of the interacting partners, and also through mutagenesis of the motifs. In each example, the base pairing rather than the absolute sequence was critical to the formation of a functional inter-segment interaction, with mutations only being tolerated in rescued virus if compensating changes were made in the interacting partner to restore uninterrupted base pairing. The absolute sequence of the complementarity motifs varied between species, indicating that this newly identified phenomenon may contribute to the observed lack of reassortment between Orbivirus species.
具有分段基因组的真核病毒选择一套完整的基因组片段包装到子代病毒颗粒中的过程尚不清楚。在本研究中，一个基于基因组片段通过碱基配对驱动的特定RNA - RNA相互作用进行关联的模型被形式化，并在呼肠孤病毒科的环状病毒属中进行了测试。在环状病毒属的模式种蓝舌病毒（BTV）中，描述了一种将基因组序列进行片段间互补性筛选与使用反向遗传学对片段间RNA - RNA相互作用进行直接功能测试相结合的策略。鉴定出了两个涉及BTV十个基因组片段中的四个的片段间特异性相互作用基序的例子，这些基序的维持对于产生感染性病毒至关重要。在所有环状病毒的直系同源基因组片段的鉴定区域之间发现了等效的片段间互补性，包括系统发育上距离较远的物种。使用电泳迁移率变动分析在体外证实了参与相互作用的RNA片段的特异性相互作用，通过使用与相互作用伙伴之一的相互作用基序互补的寡核苷酸抑制相互作用，并且也通过对基序进行诱变来实现。在每个例子中，碱基配对而非绝对序列对于形成功能性的片段间相互作用至关重要，只有在相互作用伙伴中进行补偿性改变以恢复不间断的碱基配对时，拯救出的病毒中才允许发生突变。互补基序的绝对序列在不同物种之间有所不同，这表明这种新发现的现象可能导致了环状病毒物种之间观察到的缺乏重配现象。
