In virio secondary RNA structure analysis of influenza A virus

The genome of influenza A viruses (IAV) consists of eight distinct gene segments that need to be packaged into new virions. Although this process is thought to be controlled by specific RNA-RNA interactions between segments, few studies have identified the RNA structure inside virions and performed functional validations on these structures. Using high-throughput probing techniques such as SHAPE-MaP and DMS-MaPseq, we mapped the secondary structure of the A/Puerto Rico/8/1934 (PR8) IAV genome. We discovered 173 putative structural motifs in both packaging and central regions of all segments. Fifteen motifs were selected for functional studies by introducing synonymous mutations to destabilize the predicted structure and assessing viral fitness. Synonymous mutations in three predicted RNA structures within the packaging signals of NP and PB1 attenuated virus replication in vitro. Interestingly, combining structural changes in multiple motifs within the packaging signals enhanced viral attenuation, supporting the idea of synergistic structural contributions to viral fitness. In contrast, structural synonymous mutations in RNA secondary structures outside packaging signals had no impact on virus replication in vitro. Overall, our findings revealed several new RNA secondary structures within the packaging signals of IAV gene segments that play a critical role in the IAV life cycle.