Selective assembly of influenza virus segments into virions is proposed to be mediated through intersegmental RNA-RNA interactions. Here, we developed a method called 2CIMPL that includes proximity ligation under native conditions to identify genome-wide RNA duplexes. Interactions between all eight segments were observed at multiple sites along a given segment and are concentrated at hotspots. Furthermore, synonymous nucleotide changes in a hotspot decreased the formation of RNA-RNA interactions at this site and resulted in a genome-wide rearrangement without a loss in replicative fitness. These results indicate that the viral RNA interaction network is flexible to account for nucleotide evolution. Moreover, comparative analysis of RNA-RNA interaction sites with viral nucleoprotein (NP) binding to the genome revealed that RNA junctions can also occur adjacent to NP peaks, suggesting that NP association does not exclude RNA duplex formation. Overall, 2CIMPL is a versatile technique to map in vivo RNA-RNA interactions.

提议将流感病毒区段选择性组装成病毒粒子是通过节间RNA-RNA相互作用介导的。在这里，我们开发了一种称为2CIMPL的方法，该方法包括在自然条件下进行邻近连接以鉴定全基因组RNA双链体。沿着给定路段的多个位置观察到所有八个路段之间的相互作用，并且集中在热点上。此外，热点中同义核苷酸的变化减少了该位点处RNA-RNA相互作用的形成，并导致了全基因组重排，而复制适应性没有损失。这些结果表明病毒RNA相互作用网络可以灵活地解释核苷酸进化。此外，病毒核蛋白（NP）与基因组结合后的RNA-RNA相互作用位点的比较分析表明，RNA接头也可能出现在NP峰附近，这表明NP结合并不排除RNA双链体的形成。总体而言，2CIMPL是一种通用的映射技术体内RNA-RNA相互作用。